Genes encoding acetolactate synthase

ABSTRACT

The present invention provides a gene coding for the following protein (a) or (b) showing a high level of resistance to PC herbicides or sulfonylurea herbicides: (a) a protein which consists of an amino acid sequence of any one of SEQ ID NOS: 2, 4, 6 and 8; (b) a protein which consists of an amino acid sequence derived from the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6 and 8 by substitution, deletion or addition of at least one or more amino acids, has resistance to a pyrimidinyl carboxy herbicide, and has acetolactate synthase activity.

FIELD OF THE INVENTION

The present invention relates to a gene coding for acetolactate synthasewhich is a rate-limiting enzyme in the branched-chain amino acidbiosynthetic pathway.

BACKGROUND OF THE INVENTION

Acetolactate synthase (hereinafter referred to as “ALS”) is arate-limiting enzyme in the biosynthetic pathway of branched chain aminoacids, such as leucine, valine and isoleucine, and is known as anessential enzyme for the growth of plants. ALS is also known to bepresent in a wide variety of higher plants. In addition, ALS is found invarious microorganisms, such as yeast (Saccharomyces cerevisiae),Escherichia coli, and Salmonella typhimurium.

Three types of isoenzymes of ALS are known to be present in Escherichiacoli and Salmonella typhimurium. Each of these isoenzymes is a heterooligomer consisting of catalytic subunits with a large molecular weightthat govern catalytic activity of the enzyme and regulatory subunitswith a small molecular weight that function as feedback inhibitors bybinding of branched-chain amino acids (Chipman et al., Biochim. Biophys.Acta. 1385, 401-419, 1998). Catalytic subunits are located at Ilv IH,Ilv GM and Ilv BN operons, respectively. On the other hand, ALS in yeastis a single enzyme, which comprises a catalytic subunit and a regulatorysubunit, as is the case in bacteria (Pang et al., Biochemistry, 38,5222-5231, 1999). The catalytic protein subunit is located at the locusILV2.

In plants, ALS is known to consist catalytic subunit(s) and regulatorysubunit(s) as, is the case in the above microorganisms (Hershey et al.,Plant Molecular Biology. 40, 795-806, 1999). For example, the catalyticsubunit of ALS in tobacco (dicotyledon) is coded by two gene loci, SuRAand SuRB (Lee et al., EMBO J. 7, 1241-1248, 1988); and that in maize iscoded by two gene loci, als 1 and als 2 (Burr et al., Trends in Genetics7, 55-61, 1991; Lawrence et al., Plant Mol. Biol. 18, 1185-1187, 1992).The nucleotide sequences of genes coding for a catalytic subunit havebeen completely determined for dicotyledonous plants including tobacco,Arabidopsis, rapeseed, cotton, Xanthium, Amaranthus and Kochia (seeChipman et al., Biochim. Biophys. Acta. 1385, 401-419, 1998 and domesticre-publication of PCT international publication for patent applicationsWO97/08327). However, maize and rice (Kaku et al., the 26^(th)Conference of Pesticide Science Society of Japan, Lecture Abstracts,p101, 2001) are the only monocotyledonous plants whose nucleotidesequences have been completely determined.

Meanwhile, herbicides, for example, sulfonylurea herbicides,imidazolinon herbicides, triazolopyrimidine herbicides and pyrimidinylcarboxy herbicides (hereinafter referred to as “PC herbicides”), areknown to suppress the growth of a plant by inhibiting ALS (Ray, PlantPhysiol. 75, 827-831, 1984; Shaner et al., Plant Physiol.76, 545-546,1984; Subramanian et al., Plant Physiol. 96, 310-313, 1991; Shimizu etal., J. Pestic. Sci.19, 59-67, 1994).

As shown in Tables 1 and 2, known plants having resistance to theseherbicides contain a gene coding for ALS that includes substitution ofone or two nucleotides which induces substitution of one or two aminoacids in a region conserved among different species. TABLE 1 Mutation inplant ALS which imparts resistance against ALS-inhibiting type herbicide(1) Corresponding Herbicide rice Plant species Mutation tested ALS aminoacid Zea mays Ala90Thr IM Ala96Thr Arabidopsis thaliana Ala122ValAla96Val Xantium strumarium Ala100Thr IM Ala96Thr Beta vulgarisAla113Thr IM/SU Ala96Thr Arabidopsis thaliana Met124Glu Met98GluArabidopsis thaliana Met124Ile Met98Ile Arabidopsis thaliana Met124HisMet98His Lactuca serriola Pro→His SU Pro171His Kochia scoparia Pro189ThrSU Pro171Thr Kochia scoparia Pro189Ser SU Pro171Ser Kochia scopariaPro189Arg SU Pro171Arg Kochia scoparia Pro189Leu SU Pro171Leu Kochiascoparia Pro189Gln SU Pro171Gln Kochia scoparia Pro189Ala SU Pro171AlaBrassica napus Pro173Ser Pro171Ser Nicotina tabacum Pro196Gln SUPro171Gln Nicotina tabacum Pro196Ala SU Pro171Ala Nicotina tabacumPro196Ser SU Pro171Ser Arabidopsis thaliana Pro197Ser SU Pro171SerArabidopsis thaliana Pro197deletion Pro171deletion Beta vulgarisPro188Ser IM/SU Pro171Ser Sisymbrium orientale Pro→Ile Pro171IleBrassica tournefortii Pro→Ala Pro171Ala Scirpus juncoides Pro→Leu SUPro171Leu Scirpus juncoides Pro179Ala SU Pro171Ala Scirpus juncoidesPro179Gln SU Pro171Gln Scirpus juncoides Pro179Ser SU Pro171Ser Scirpusjuncoides Pro179Lys SU Pro171Lys Lindernia micrantha Pro→Gln SUPro171Gln Lindernia procumbens Pro→Ser SU Pro171Ser Lindernia dubiasubsp. Pro→Ser SU Pro171Ser Lindernia dubia Pro→Ala SU Pro171AlaArabidopsis thaliana Arg199Ala Arg173Ala Arabidopsis thaliana Arg199GluArg173Glu Xantium strumarium Ala183Val Ala179Val Arabidopsis thalianaPhe206Arg Phe180Arg

TABLE 2 Mutation in plant ALS which imparts resistance to ALS-inhibitingtype herbicide (2) Corresponding Herbicide rice Plant species Mutationtested ALS amino acid Kochia scoparia Asp260Gly SU Asp242Gly Kochiascoparia Trp487Arg SU Try465Arg Kochia scoparia Asn561Ser SU Asn539SerKochia scoparia Trp570Leu Trp548Leu Gossypium hirsutum L. Trp563Cys SU ?Try548Cys Gossypium hirsutum L. Trp563Ser SU ? Try548Ser Brassica napusTrp557Leu Try548Leu Zea mays L. Trp552Leu IM Try548Leu Nicotina tabacumL. Trp537Leu SU Try548Leu Arabidopsis thaliana Trp574Leu Try548LeuArabidopsis thaliana Trp574Ser Try548Ser Arabidopsis thalianaTrp574deletion Try548deletion Xantium strumarium Trp552Leu IM Try548LeuOryza sativa. Trp548Leu PC Try548Leu Amaranthus sp. Trp569Leu Try548LeuAmaransus rudis Trp569Leu IMI Try548Leu Sisymbrium orientale Trp→LeuTry548Leu Zea mays Ser621Asp IM Ser627Asp Zea mays Ser621Asn IMSer627Asn Arabidopsis thaliana Ser653Asn IM Ser627Asn Arabidopsisthaliana Ser653Thr Ser627Thr Arabidopsis thaliana Ser653Phe Ser627PheArabidopsis thaliana Ser653delition Ser627deletion Oryza sativaSer627Ile PC Ser627Ile Kochia scoparia Val276Glu SU

Examples of such a gene include a gene coding for ALS having resistancespecific to sulfonylurea herbicides (see Kathleen et al., EMBO J. 7,1241-1248, 1988; Mourad et al., Planta, 188, 491-497, 1992; Guttieri etal., Weed Sci. 43, 175-178, 1995; Bernasconi et al., J. Biol. Chem. 270,17381-17385, 1995; and JP Patent Publication (Unexamined Application)No. 63-71184); a gene coding for ALS having resistance specific toimidazolinon herbicides (see Mourad et al., Planta, 188, 491-497, 1992;Lee et al., FEBS Lett. 452, 341-345, 1999; and JP Patent Publication(Unexamined Application) No. 5-227964); a gene coding for ALS havingresistance to both sulfonylurea and imidazolinon herbicides (seeKathleen et al., EMBO J. 7, 1241-1248, 1988; Bernasconi et al., J. Biol.Chem. 270, 17381-17385, 1995; Hattori et al., Mol. Gen. Genet. 246,419-425, 1995; Alison et al., Plant Physiol. 111, 1353, 1996;Rajasekarau et al., Plant Sci. 119, 115-124, 1996; JP Patent Publication(Unexamined Application) No. 63-71184; JP Patent Publication (UnexaminedApplication) No. 4-311392; and Bernasconi et al., U.S. Pat. No.5,633,437, 1997); and a gene coding for ALS having a high level ofresistance to PC herbicides (Kaku et al., the 26^(th) Conference ofPesticide Science Society of Japan, Lecture Abstracts, p101, 2001). Theproduction of a plant body showing resistance to both sulfonylurea andimidazolinon herbicides has been attempted by crossing a plant havingALS showing resistance specific to sulfonylurea herbicides with a planthaving ALS showing resistance specific to imidazolinon herbicides(Mourad et al., Mol. Gen. Genet, 243, 178-184, 1994). Furthermore,artificial alteration of a gene coding for ALS into a herbicideresistance gene has been attempted (see Ott et al., J. Mol. Biol. 263,359-368, 1996, JP Patent Publication (Unexamined Application) No.63-71184, JP Patent Publication (Unexamined Application) No. 5-227964,JP Patent Publication (PCT Translation) No. 11-504213), such that it hasbeen found that a single amino acid deletion causes ALS to showresistance to both sulfonylurea and imidazolinon herbicides (see JPPatent Publication (Unexamined Application) No. 5-227964).

As described above. ALSs having resistance to herbicides; and genescoding for ALS have been aggressively studied. However, only a few caseshave been reported concerning a mutant ALS gene having resistancespecific to a PC herbicide using resistance to PC herbicides as anindicator. Moreover, there have been also only a few cases reportedconcerning the study of the resistance to PC herbicides and otherherbicides.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a gene coding for anALS protein showing extremely high level of resistance to PC herbicidesor to sulfonylurea herbicides, an ALS protein coded by the gene, arecombinant vector having the gene, a transformant having therecombinant vector, a plant having the gene, a method for rearing theplant, and a method for selecting a transformant cell using the gene asa selection marker.

As a result of thorough studies to achieve the above purpose, we havecompleted the present invention by finding that a mutant ALS which isderived from the wild type ALS by substituting a certain amino acidresidue of the wild type ALS with a certain amino acid shows extremelyhigh resistance to PC herbicides.

(1) Specifically, the present invention is a gene which codes for thefollowing protein (a) or (b):

-   -   (a) a protein consisting of an amino acid sequence of any one of        SEQ ID NOS: 2, 4, 6 and 8;    -   (b) a protein consisting of an amino acid sequence derived from        the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6 and 8        by substitution, deletion or addition of at least one or more        amino acids, which has resistance to PC herbicides and has        acetolactate synthase activity.

(2) Further, the present invention is an acetolactate synthase protein,which is coded by the gene of (1).

(3) Furthermore, the present invention is a recombinant vector, whichhas the gene of (1).

(4) Further, the present invention is a transformant, which has therecombinant vector of (3).

(5) Moreover, the present invention is a plant, which has the gene of(I) and has resistance to PC herbicides.

(6) Further, the present invention is a method for cultivating the plantof (5) which comprises cultivating the plant in the presence of a PCherbicide.

(7) Still further, the present invention is a method for selecting atransformant cell having the gene of (1), which uses this gene as aselection marker.

Hereunder, a more detailed explanation will be given of the presentinvention.

The gene coding for the acetolactate synthase of the present invention(hereinafter referred to as “mutant ALS gene”) codes for an acetolactatesynthase protein (hereinafter referred to as “mutant ALS protein”)having an amino acid sequence that is different from that of a wild typeacetolactate synthase protein (hereinafter, referred to as “wild typeALS protein”). The mutant ALS protein can be obtained by mutating acertain site in a wild type ALS protein expressed in a rice plant. Themutant ALS protein of the present invention consists of the amino acidsequence of any one of SEQ ID NOS: 2, 4, 6, and 8.

The amino acid sequence of SEQ ID NO: 2 is derived from the amino acidsequence of the wild type ALS protein by substitution of proline 171with histidine and substitution of arginine 172 with serine. A mutantALS protein containing the amino acid sequence of SEQ ID NO: 2 isreferred to as “P171H/R172S mutant ALS protein,” or “P171H/R172Smutant.”

The amino acid sequence of SEQ ID NO: 4 is derived from the amino acidsequence of the wild type ALS protein by substitution of proline 171with histidine and substitution of tryptophan 548 with leucine. A mutantALS protein containing the amino acid sequence of SEQ ID NO: 4 isreferred to as “P171H/W548L mutant ALS protein,” or “P171H/W548Lmutant.”

The amino acid sequence of SEQ ID NO: 6 is derived from the amino acidsequence of the wild type ALS protein by substitution of proline 171with histidine, and substitution of serine 627 with isoleucine. A mutantALS protein containing the amino acid sequence of SEQ ID NO: 6 isreferred to as “P171H/S627I mutant ALS protein,” or “P 171H/S627Imutant.”

The amino acid sequence of SEQ ID NO: 8 is derived from the amino acidsequence of the wild type ALS protein by substitution of proline 171with histidine, substitution of tryptophan 548 with leucine, andsubstitution of serine 627 with isoleucine. A mutant ALS proteincontaining the amino acid sequence of SEQ ID NO: 8 is referred to as“P171H/W548L/S627I mutant ALS protein,” or “P171H/W548L/S627I mutant.”

FIGS. 1A and B show the results of comparisons among the amino acidsequences of these 4 types of mutant ALS proteins and the amino acidsequence of the wild type ALS protein. Further, in FIGS. 1A and B, theamino acid sequence in the 1^(st) row represents the wild type ALSprotein, the amino acid sequence in the 2^(nd) row representsP171H/R172S mutant ALS protein, the amino acid sequence in the 3^(rd)row represents P171H/W548L mutant ALS protein, the amino acid sequencein the 4^(th) row represents P171H/S627I mutant ALS protein, and theamino acid sequence in the 5^(th) row represents P171H/W548L/S627Imutant ALS protein.

Compared to the wild type ALS protein, these mutant ALS proteins possessgood resistance not only to PC herbicides, but also to sulfonylurea andimidazolinon herbicides. This can be determined by subcloning a genecoding for the mutant ALS protein into pGEX 2T, transforming E. coli orthe like with the pGEX 2T, and then examining the sensitivity of theexpressed mutant ALS protein to herbicides.

Examples of a PC herbicide include bispyribac-sodium, pyrithiobac-sodiumand pyriminobac, as represented by the following chemical formula 1.

An example of a sulfonylurea herbicide is chlorsulfuron, as representedby the following chemical formula 2.

An example of an imidazolinon herbicide is imazaquin, as represented bythe following chemical formula 3.

In particular, P171H/R172S mutant ALS protein shows resistance to acertain herbicide at a level not only better than that of a mutant ALSprotein independently having P171H or R172S, but also superior to thecombined resistance predicted from the mutant ALS proteins independentlyhaving P171H or R172S. Further, the mutant ALS protein independentlyhaving R172S does not show resistance to any herbicides, therefore theR172S mutation is a silent mutation. In other words, in P171H/R172Smutant ALS protein, R172S mutation, which is a silent mutation byitself, improves the resistance of P171H mutant ALS protein.

Further, P171H/W548L mutant protein shows resistance to a certainherbicide at a level not only better than that of a mutant ALS proteinindependently having P171H or W548L, but also better than the combinedresistance predicted from the mutant ALS proteins independently havingP171H or W548L. In other words, P171H/W548L mutant protein showsresistance which is far greater than the synergistic effect predictedfrom the resistances of both P171H mutant protein and W548L mutantprotein.

Further, in particular, P171H/S627I mutant protein shows resistance to acertain herbicide at a level not only better than that of a mutant ALSprotein independently having P171H or S627I, but also better than thecombined resistance predicted from the mutant ALS proteins independentlyhaving P171H or S627I. In other words, P171H/S627I mutant protein showsresistance which is far greater than the synergistic effect predictedfrom the resistances of both P171H mutant protein and S627I mutantprotein.

Still further, in particular, P171H/W548L/S627I mutant protein showsresistance to a certain herbicide better than that of a mutant ALSprotein independently having P171H, W548L or S627I.

Moreover, the mutant ALS protein of the present invention may consist ofany amino acid sequence derived from the amino acid sequence of any oneof SEQ ID NOS: 2, 4, 6 and 8 by substitution, deletion or addition of atleast one or more amino acids, as long as the sequence has resistance toa PC herbicide and has acetolactate synthase activity. Here, “one ormore amino acids” preferably refers to 1 to 30 amino acids, morepreferably 1 to 20 amino acids, and more preferably 1 to 10 amino acids.

Particularly, in the amino acid sequence of SEQ ID NO: 2, “at least oneor more amino acids” are preferably (an) amino acids other than the171^(st) and 172^(nd) amino acids. In the amino acid sequence of SEQ IDNO: 4, “at least one or more amino acids” are preferably (an) aminoacids other than the 171^(st) and 548^(th) amino acids. In the aminoacid sequence of SEQ ID NO: 6, “at least one or more amino acids” arepreferably (an) amino acids other than the 171^(st) and 627^(th) aminoacids. In the amino acid sequence of SEQ ID NO: 8, “at least one or moreamino acids” are preferably (an) amino acids other than the 171^(st),627^(th), and 548^(th) amino acids.

The mutant ALS gene of the present invention is not specificallylimited, as long as it has a nucleotide sequence coding for theabove-described mutant ALS protein. Examples of the nucleotide sequenceinclude the nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5 and 7.The nucleotide sequence of SEQ ID NO: 1 codes for the amino acidsequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 3 codesfor the amino acid sequence of SEQ ID NO: 4, the nucleotide sequence ofSEQ ID NO: 5 codes for the amino acid sequence of SEQ ID NO: 6, and thenucleotide sequence of SEQ ID NO: 7 codes for the amino acid sequence ofSEQ ID NO: 8. The mutant ALS gene may have a nucleotide sequence derivedfrom the nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5 and 7 bysubstitution of a codon coding for a certain amino acid with adegenerate codon.

FIGS. 2A, B, C and D show the results of comparisons among thenucleotide sequences coding for these 4 types of mutant ALS proteins andthe nucleotide sequence coding for a wild type ALS protein. In FIGS. 2A,B, C and D, the nucleotide sequence in the 1^(st) row represents thewild type ALS protein, the nucleotide sequence in the 2^(nd) rowrepresents P171H/R172S mutant ALS protein, the nucleotide sequence inthe 3^(rd) row represents P171H/W548L mutant ALS protein, the nucleotidesequence in the 4^(th) row represents P171H/S627I mutant ALS protein,and the nucleotide sequence in the 5^(th) row representsP171H/W548L/S627I mutant ALS protein.

Moreover, the mutant ALS gene of the present invention may consist of anucleotide sequence which can hybridize under stringent conditions to anucleotide sequence complementary to the nucleotide sequence of any oneof SEQ ID NOS: 1, 3, 5 and 7, and codes for an amino acid sequencehaving acetolactate synthase activity. “Stringent conditions” refers toconditions wherein a so-called specific hybrid is formed and anon-specific hybrid is not formed. Examples of such stringent conditionsinclude conditions whereby DNAs having high homology to each other (forexample, DNAs having 50% or more homology to each other) hybridize andDNAs having low homology to each other do not hybridize. Specificexamples of the stringent conditions, under which hybridization ispossible, include conditions for washing in the normal Southernhybridization of 60° C., and a salt concentration corresponding to1×SSC, 0.1% SDS, or preferably, 0.1×SSC, 0.1% SDS.

Genes coding for these mutant ALS proteins can be obtained byintroducing a mutation as described above into a gene coding for a wildtype ALS protein which is present in the genomic DNA of japonica typerice variety, Kinmaze. To introduce mutations, any known techniques canbe employed. For example, site-directed mutagenesis can be used.Site-directed mutagenesis can be performed using a commercial kit, e.g.,Mutan-K (Takara Shuzo), Gene Editor (Promega) or ExSite (Stratagene).

In addition, a gene coding for the mutant ALS protein can be obtained byculturing wild type culture cells sensitive to a PC herbicide in thepresence of the PC herbicide and then obtaining the gene from mutantculture cells that appear and show resistance to the PC herbicide. Then,a gene coding for ALS protein having a new combination of mutations canbe synthesized based on the thus found mutations by the PCR method andSPR (self polymerase reaction) method using enzymes.

Specifically, first, mRNAs are prepared from mutant culture cellsresistant to a PC herbicide, cDNAs are synthesized, and then a cDNAlibrary of λgt 11 phage is constructed. Then, the library is screenedusing a nucleic acid probe containing part of a gene coding for the wildtype ALS protein. Next, the insert DNA of the resulting positive cloneis subcloned into pBluescript II SK+, to determine the nucleotidesequence. For cDNA inserts that have been shown to have mutations,fragments containing the mutation are synthesized by the PCR and SPRmethods using as a template pBluescript II SK+ retaining the insert DNA,and primers designed based on the wild type rice ALS gene. Meanwhile,genomic DNAs are prepared from PC-herbicide-resistant rice culturecells, and various primers are designed based on rice ALS genes. Then,primer walking is performed, so that the sequences of ALS genes presentin the prepared genomic DNAs are determined, and mutations sites arefound. When mutations are found, fragments containing the mutations aresynthesized by the PCR and SPR methods. Fragments containing mutationssynthesized from mutant ALS cDNA cloned into pBluescript II SK+(including the fragments containing these mutations) are subcloned intopGEX 2T, and then E. coli is transformed using the vector.

Clones having the insert DNAs coding for the amino acid sequencesrepresented by SEQ ID NOS: 2, 4, 6 or 8 are then selected, so that genescoding for mutant ALS proteins can be obtained. In addition, the thusobtained plasmid in which a gene coding for a mutant ALS proteincontaining the amino acid sequence represented by SEQ ID NO: 2 had beenincorporated in pGEX 2T was deposited as Rice Mutant ALS cDNA 1 (FERMBP-7944), the plasmid in which a gene coding for a mutant ALS proteincontaining the amino acid sequence represented by SEQ ID NO: 4 had beenincorporated in pGEX 2T was deposited as Rice Mutant ALS cDNA 2 (FERMBP-7945), the plasmid in which a gene coding for a mutant ALS proteincontaining the amino acid sequence represented by SEQ ID NO: 6 had beenincorporated in pGEX 2T was deposited as Rice Mutant ALS cDNA 3 (FERMBP-7946), and the plasmid in which a gene coding for a mutant ALSprotein containing the amino acid sequence represented by SEQ ID NO: 8had been incorporated in pGEX 2T was deposited as Rice Mutant ALS cDNA 4(FERM BP-7947) with the Patent and Bio-Resource Center, NationalInstitute of Advanced Industrial Science and Technology (Chuo-6, 1-1-1,Higashi, Tsukuba-shi, Ibaraki, JAPAN) on Mar. 8, 2002 under the BudapestTreaty.

On the other hand, transformation of a target plant using a gene codingfor the mutant ALS protein can impart resistance to various herbicides,such as PC herbicides, to the plant. Any known technique can be used fortransformation of a plant. For example, a foreign gene can be introducedinto a target plant cell using Agrobacterium tumefaciens.

More specifically, a gene coding for the mutant ALS protein is insertedinto a binary vector containing T-DNA sequence of a Ti plasmid ofAgrobacterium. The Ti plasmid is transformed into E. coli and the like.Then, the binary vectors retaining the gene coding for the mutant ALSprotein replicated by, e.g., E. coli are transformed into Agrobacteriawhich contain helper plasmids. Target plants are infected with theAgrobacteria, and then the transformed plants are identified. When theidentified transformed plant is a culture cell, the plant cell can beregenerated into a complete plant by any known technique.

To transform a target plant with a gene coding for the mutant ALSprotein, the gene can be directly introduced using known standardtechniques. Examples of a method which transforms an expression vectorcontaining a gene coding for the mutant ALS protein include thepolyethylene glycol method, electroporation, and the particle gunmethod.

A gene coding for the mutant ALS protein may be transformed into anytype of plant, such as monocotyledonous and dicotyledonous plants.Examples of a target crop into which a gene coding for the mutant ALSprotein is transformed include rice, maize, wheat, barley, soybean,cotton, rapeseeds, sugar beet and tobacco. In addition, turf grass,trees and the like can be transformed by introducing a gene coding forthe mutant ALS protein.

In any of the above cases, transformation of a plant using a gene codingfor the mutant ALS protein can impart resistance to PC herbicides,sulfonylurea herbicides, and imidazolinon herbicides to the plant.

Moreover, a gene coding for the mutant ALS protein can also be used as aselection marker in an experiment for transformation of a plant. Forexample, to transform a plant cell using a target gene, a vector whichhas a gene coding for the mutant ALS protein and a target gene isintroduced into the plant cell, followed by culturing of the plant cellunder the presence of a PC herbicide or the like. If a plant cellsurvives in the presence of the herbicide, it indicates that the plantcell contains a gene coding for the mutant ALS protein and the gene ofinterest introduced therein. Further, whether a target gene and a genecoding for the mutant ALS protein are incorporated into the chromosomeof a plant cell can be confirmed by observing the phenotype of the plantand then examining the presence of these genes on the genome, by genomesouthern hybridization or PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an amino acid sequence comparison between the mutant ALSproteins and the wild type ALS protein.

FIG. 1B is a continuation from FIG. 1A, and shows an amino acid sequencecomparison between the mutant ALS proteins and the wild type ALSprotein.

FIG. 2A shows a nucleotide sequence comparison between the mutant ALSgenes and the wild type ALS gene.

FIG. 2B is a continuation from FIG. 2A, and shows a nucleotide sequencecomparison between the mutant ALS genes and the wild type ALS gene.

FIG. 2C is a continuation from FIG. 2B, and shows a nucleotide sequencecomparison between the mutant ALS genes and the wild type ALS gene.

FIG. 2D is a continuation from FIG. 2C, and shows a nucleotide sequencecomparison between the mutant ALS genes and the wild type ALS gene.

FIG. 3 is a characteristic figure showing sensitivity of Rb line tobispyribac-sodium.

FIG. 4 is a characteristic figure showing sensitivity of Sr line tobispyribac-sodium.

FIG. 5 is a characteristic figure showing sensitivity of Ga line tobispyribac-sodium.

FIG. 6 is a characteristic figure showing sensitivity of Vg line tobispyribac-sodium.

FIG. 7 is a characteristic figure showing sensitivity of the wild typeto bispyribac-sodium.

FIG. 8 is a characteristic figure showing sensitivity of the wild typeto chlorsulfuron.

FIG. 9 is a characteristic figure showing sensitivity of Rb line tochlorsulfuron.

FIG. 10 is a characteristic figure showing sensitivity of Sr line tochlorsulfuron.

FIG. 11 is a characteristic figure showing sensitivity of Ga line tochlorsulfuron.

FIG. 12 is a characteristic figure showing sensitivity of Vg line tochlorsulfuron.

FIG. 13 is a characteristic figure showing the relation between thefraction number and absorbance at OD 525 nm in anion exchange columnchromatography performed for the purpose of separating the ALS proteinof the resistant mutant.

FIG. 14 is a characteristic figure showing the relation between thefraction number and absorbance at OD 525 nm in anion exchange columnchromatography performed for the purpose of separating the wild type ALSprotein.

FIG. 15 is a characteristic figure showing sensitivity of the wild typeALS protein and the mutant ALS protein to bispyribac-sodium.

FIG. 16 is a characteristic figure showing sensitivity of the wild typeALS protein and the mutant ALS protein to chlorsulfuron.

FIG. 17 is a characteristic figure showing sensitivity of the wild typeALS protein and the mutant ALS protein to imazaquin.

FIG. 18A shows a nucleotide sequence comparison between Nippon-bare ESTand maize ALS gene.

FIG. 18B is a continuation from FIG. 18A and shows a nucleotide sequencecomparison between Nippon-bare EST and maize ALS gene.

FIG. 19A is a nucleotide sequence comparison between the full-lengthcDNA derived from Sr line and wild type cDNA 1.

FIG. 19B is a continuation from FIG. 19A, and shows a nucleotidesequence comparison between the full-length cDNA derived from Sr lineand wild type cDNA 1.

FIG. 19C is a continuation from FIG. 19B, and shows a nucleotidesequence comparison between the full-length cDNA derived from Sr lineand wild type cDNA 1.

FIG. 20 shows processes for synthesizing ALS cDNAs independently havingG1643T (W548L) mutation or G1880T (S627I) mutation, and for constructingpGEX 2T retaining the ALS cDNA. Arrows denote primers, and asterisksdenote mutated points.

FIG. 21 shows a process for preparing C512A (P171H) mutant DNA fragmentand C514A (R172S) mutant DNA fragment. Arrows denote primers, andasterisks denote mutated points.

FIG. 22 shows processes for synthesizing ALS cDNAs independently havingC512A (P171H) mutation or C514A (R172S) mutation, and for constructingpGEX 2T retaining the ALS cDNA. Asterisks denote mutated points.

FIG. 23 shows a process for preparing a DNA fragment havingC512A(P171H)/C514A(R172S). Arrows denote primers, and asterisks denotemutated points.

FIG. 24 shows processes for synthesizing P171H/W548L mutant ALS cDNA andP171H/S627I mutant ALS cDNA and for constructing pGEX 2T retaining theALS cDNA. Asterisks denote mutated points.

FIG. 25 shows processes for synthesizing P171H/W548L/S627I mutant ALScDNA and for constructing pGEX 2T retaining the ALS cDNA. Asterisksdenote mutated points.

FIG. 26 shows a comparison of sensitivity to bispyribac-sodium betweenthe mutant ALS protein coded by 1-point mutant ALS gene and the wildtype ALS protein.

FIG. 27 shows a comparison of sensitivity to bispyribac-sodium among themutant ALS proteins coded by 2-point and 3-point mutant ALS genes andthe wild type.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, the present invention will be further described by the followingexamples, but the technical scope of the invention is not limited bythese examples.

EXAMPLE 1 Production of Rice (Kinmaze) Culture Cells Resistant to a PCHerbicide

Chaff was removed from rice seeds (variety: Kinmaze, scientific name:Oryza saliva var. Kinmaze). The seeds were immersed in 70% ethanol for 5minutes, and then immersed in about 5% antiformin for 20 minutes,followed by washing several times with sterile distilled water. Then,the seeds were static-cultured on a medium with a composition as shownin Table 3. TABLE 3 Inorganic salt (mixed saline for  1 packMurashige-Skoog medium) Thiamin·HCl (0.1 g/l)  1 ml Nicotinic acid (0.5g/l)  1 ml Pyridoxine·HCl (0.5 g/l)  1 ml Glycine (2 g/l)  1 mlmyo-inositol (50 g/l)  2 ml 2,4-D (200 ppm) 10 ml Sucrose 30 g Gelrite 3 g Prepare the medium to 1000 ml with distilled water, and adjust pHto 5.7.

In the above medium composition, 2,4-D is synthesized auxin. To preparethe medium, first, a medium with the above composition was placed in a11 beaker, and distilled water was added to the beaker to 1000 ml. Next,the solution was adjusted to pH 5.7, and supplemented with 3 g ofGelrite. The Gelrite was dissolved well by heating with a microwaveoven, and then the mixture was added 30 ml at a time to culture flasksusing a pipetter. Next, three sheets of aluminum foil were laid over theculture flask, followed by heating for sterilization in an autoclave at121° C. for 15 to 20 minutes. Finally the solution was cooled to roomtemperature so that the media for static culture of the above seeds wereprepared.

Next, endosperm portions were removed from the callus induced on themedium, and then subculture was performed. Then, part of the obtainedcalli was sub-cultured, that is, cultured successively once per twoweeks in a liquid medium (the composition is the same as in that shownin Table 3, but not supplemented with Gelrite) supplemented with 1 μMbispyribac-sodium (one type of PC herbicides). Two to 6 weeks later theculture cells started to wither. About 2 months later, a plurality ofnon-discolored cell masses that were thought to be conducting celldivision were obtained from among culture cell populations most of whichhad died and became discolored brown. These cell masses were isolatedand cultured, so that a plurality of cell lines that can proliferate inthe presence of 2 μM bispyribac-sodium were obtained. The obtained celllines were named Rb line, Sr line, Ga line and Vg line, respectively.

Subsequently, the resulting plurality of cell lines were cultured whileelevating the concentration of bispyribac-sodium in an orderly manner.As a result, cell lines that can proliferate in the presence of 100 μMbispyribac-sodium were obtained. The bispyribac-sodium resistant culturecells (hereinafter referred to as “resistant mutant”) were sub-culturedon MS-2,4-D solid media supplemented with 1 to 10 μM bispyribac-sodium.Part of the sub-cultured resistant mutant was sampled, added intoMS-2,4-D liquid media not supplemented with bispyribac-sodium, and thensubjected to suspended cell culture at a cycle of 8 to 10 days.

Approximately 1.5 g (wet weight) of the resistant mutant wastransplanted into a 200 ml Erlenmeyer flask supplemented with 50 ml of aMS-2,4-D liquid medium and bispyribac-sodium at a certain concentration,followed by culturing at approximately 27° C. for a certain period. Thewet weight of the callus was measured periodically. The relative amountof increase was determined based on the wet weight of the transplantedresistant mutant. In addition, the experiment was performed three timeswith different bispyribac-sodium concentrations, and the standard errorwas calculated. FIGS. 3 to 6 show the relation between changes inbispyribac-sodium concentration and the relative weight on day 8 or 12in the resistant mutant. As a control, a similar experiment wasconducted using the wild type (Kinmaze). FIG. 7 shows the result ofmeasuring the relation between bispyribac-sodium concentration andrelative weight on day 8 in the wild type (Kinmaze).

As shown in FIG. 7, the growth of the wild type was not inhibited in agroup supplemented with 1 nM bispyribac-sodium, but was inhibited in agroup supplemented with 10 nM or more bispyribac-sodium. On the otherhand, as shown in FIGS. 3 to 6, almost none of the growth of theresistant mutants (Rb line, Sr line, Ga line, and Vg line) other than Vgline was affected even in a group supplemented with 10 μMbispyribac-sodium. Even in Vg line, it was shown that the effect ofbispyribac-sodium on the growth was smaller than that in the wild type.

Also in the case of using chlorsulfuron instead of bispyribac-sodium,the growth rates of the wild type and the resistant mutants weremeasured as described above. FIG. 8 shows the relation between changesin chlorsulfuron concentration and relative weight on day 9 in the wildtype. Further, FIGS. 9 to 12 show the relation between changes inchlorsulfuron concentration and relative weight on day 8 or 10 in theresistant mutants, that is, Rb line, Sr line, Ga line and Vg line.

As shown in FIG. 8, the growth of the wild type was inhibited byaddition of 1 nM chlorsulfuron, showing that the wild type has highersensitivity to chlorsulfuron than to bispyribac-sodium. However, asshown in FIGS. 9 to 12, Rb line, Sr line, Ga line and Vg line differedin sensitivity, but the growth was not inhibited so much by addition ofchlorsulfuron, showing their resistance to chlorsulfuron. Sensitivity tobispyribac-sodium and chlorsulfuron remained almost unchanged in boththe wild type and the resistant mutants, even with longer cultureduration. The growth rate was almost the same in the wild type and theresistant mutants.

These results revealed that the resistant mutants possess highresistance to bispyribac-sodium. Moreover, the resistant mutants wereshown to have improved resistance to chlorsulfuron compared to the wildtype.

EXAMPLE 2 Herbicide Sensitivity of ALS Protein Partially Purified fromthe Resistant Mutant

In this example, mutant ALS protein was partially purified from theresistant mutants obtained in Example 1 (Rb line, Sr line and Vg line,with Ga line excluded), and then herbicide sensitivity of the obtainedmutant ALS protein was examined. The mutant ALS protein was partiallypurified as follows.

First, 200 g or more of resistant mutant was prepared by a liquidculture method (no supplementation with bispyribac-sodium), using acomposition as shown in Table 3 excluding Gelrite. Then, about 150 g ofthe resistant mutant was homogenized using Hiscotron in a volume ofbuffer-1 [100 mM potassium phosphate buffer (pH 7.5) containing 20%(v/v) glycerol, 0.5 mM thiamin pyrophosphate (TPP), 10 μM flavin adeninedinucleotide (FAD), 0.5 mM MgCl₂, and a volume of polyvinylpolypyrrolidone one-tenth that of tissue volume] 3-fold greater thantissue volume. The homogenate was filtered through nylon gauze, and thencentrifuged at 15000×g for 20 minutes. Ammonium sulfate was added to thecentrifuged supernatant to 50% saturation, and then allowed to stand inice for approximately 1 hour. The mixture was again centrifuged at15000×g for 20 minutes, and then the precipitated fraction was dissolvedin approximately 30 ml of buffer-2 [10 mM Tris hydrochloric acid buffer(pH 7.5) containing 20% (v/v) glycerol, 0.5 mM TPP and 0.5 mM MgCl₂].The mixture was again centrifuged at 15000×g for 20 minutes, and thenthe supernatant fraction was applied to a Sephadex G-25 (AmershamBioscience). About 40 ml of the fraction that had passed through thecolumn was collected as a crude enzyme solution.

Next, the protein concentration of the crude enzyme solution wasmeasured by the Bradford method according to the manual of Bio-RadProtein Assay. The crude enzyme solution was then filtered through aWhatman filter (Whatman), and then the crude enzyme solution in anappropriate protein amount (10 to 15 ml) was applied to threevertically-connected HiTrap Q columns (Amersham Bioscience) using a FPLCdevice (Amersham Bioscience). After protein component was adsorbed usingHiTrap Q, unadsorbed fractions were washed out using buffer-2 having avolume 3 to 5 fold greater than the bed volume. Then, the adsorbedprotein component was eluted using an eluate having a volume 10 foldgreater than the bed volume (150 ml). Here, the eluate was prepared bydissolving KCl with a linear concentration gradient (0 to 0.4 M) intobuffer-2. The eluate containing the eluted protein component wasapportioned, 5 ml each, into a plurality of test tubes for apportioning.Further, to stabilize ALS protein contained in the eluted proteincomponent, 0.5 ml of buffer-2 containing 20 mM sodium pyruvate had beenpreviously added to each test tube for apportioning.

ALS activity resulting from the mutant ALS protein contained in theeluted fractions apportioned into each test tube for apportioning wasmeasured as follows. A reaction solution to be used in a measurementreaction was prepared by mixing an eluted fraction to be measured with asolution comprising 20 mM sodium pyruvate, 0.5 mM TPP, 0.5 mM MgCl₂, 10μM FAD and 20 mM potassium phosphate buffer (pH 7.5). One ml of thisreaction solution was used. After the eluted fraction to be measured wasadded, the measurement reaction was performed at 30° C. for 40 to 60minutes. Then, the reaction was stopped by addition of 0.1 ml of 6Nsulfuric acid (or 0.25 N sodium hydroxide).

After the reaction was stopped, the reaction solution was incubated at60° C. for 10 minutes, thereby converting acetolactate contained in thereaction solution to acetoin.

Then, to quantify acetoin contained in the reaction solution, 1 ml of0.5% (w/v) creatine and 1 ml of 5% (w/v) α-naphthol dissolved in 2.5 Nsodium hydroxide was added to the reaction solution, followed byincubation at 37° C. for 10 minutes. Acetoin was then quantified bycolor comparison of the absorbance (at 525 nm) of the reaction solution,thereby evaluating ALS activity. In addition, since the reactionsolution contained a small amount of sodium pyruvate, reaction time 0was used as control.

As a result, absorbance at OD525 nm was as high as approximately 7 per0.2 ml of the reaction solution. However, when the above measurementreaction was ceased with sodium hydroxide, and acetoin generationactivity due to activity other than ALS activity was examined, nearly80% of the apparent ALS activity resulted from direct acetoin generationactivity which was not due to activity of the mutant ALS protein.Accordingly, the mutant ALS protein and the other proteins wereseparated for acetoin generation activity by FPLC using anion exchangeresin. FIG. 13 shows the result in the case of using Sr line as aresistant mutant. As a result, three activity peaks were detected asshown in FIG. 13.

To determine which one of the three activity peaks corresponded to themutant ALS protein, acetoin generation activity was examined for eachpeak. Thus it was found that a fraction shown by the peak of initialelution corresponded to the mutant ALS protein.

Using the enzyme solution containing the mutant ALS protein, sensitivityof the mutant ALS protein to bispyribac-sodium, chlorsulfuron andimazaquin was examined. Sensitivity to each of these herbicides wasevaluated by measuring ALS activity in the same manner as in the abovemeasurement reaction, except that a herbicide was added to a certainconcentration before addition of the enzyme solution. For comparison,the wild type ALS protein was separated and purified (FIG. 14) in thesame manner and used for the experiment. In addition, bispyribac-sodiumwas prepared as an aqueous solution, and chlorsulfuron and imazaquinwere prepared as acetone solutions. The final concentration of acetonein the reaction mixture was 1%.

FIG. 15 shows the relation between ALS activity inhibition rate andbispyribac-sodium concentration. FIG. 16 shows the relation between ALSactivity inhibition rate and chlorsulfuron concentration. FIG. 17 showsthe relation between ALS activity inhibition rate and imazaquinconcentration. In these FIGS. 15 to 17, a dotted line denotes the wildtype ALS protein, a long dashed double-dotted line denotes Sr line ofthe mutant ALS protein, a solid line denotes Rb line of the mutant ALSprotein, and a long dashed dotted line denotes Vg line of the mutant ALSprotein.

A herbicide concentration which inhibits 50% of ALS activity (150) wasfound from calculation according to probit analysis, thereby calculatingthe ratio of 150 for the mutant ALS protein vs. 150 for the wild typeALS protein. Table 4 shows the results. TABLE 4 I₅₀ (nM) Herbicide Wildtype Vg Sr Rb Bispyribac-sodium 5.63 97.2 421 247000 Chlorsulfuron 17.3495 92.8 32000 Imazaquin 1480 44100 16700 609000

Further, based on the results in Table 4, 150 of the resistant mutantagainst each herbicide was divided by 150 of the wild type to work outRS. The results are shown in Table 5. TABLE 5 RS ratio Herbicide Vg SrRb Bispyribac-sodium 17.3 74.8 43900 Chlorsulfuron 28.6 5.36 1850Imazaquin 29.8 11.3 411

As shown in FIGS. 15 to 17 and Tables 4 and 5, the mutant ALS proteinshowed a relatively high ALS activity even in the presence of theherbicide, when compared to the wild type ALS protein. In particular,the mutant ALS proteins derived from Rb line and Sr line were shown tohave sensitivity to bispyribac-sodium which was significantly superiorto sensitivities to other herbicides. That is, Rb and Sr lines possessgood resistance to bispyribac-sodium in particular.

EXAMPLE 3 Cloning of Wild Type and Mutant ALS Genes

In this example, a gene (wild type ALS gene) coding for the wild typeALS protein was cloned from the wild type, while a gene (mutant ALSgene) coding for the mutant ALS protein was cloned from the resistantmutant.

Probes used for cloning the wild type ALS gene and the mutant ALS genewere prepared as follows. The partial cDNA derived from rice(Nippon-bare) showing high homology with the ALS gene of maize was usedas a probe in this example.

(1) Determination of the Nucleotide Sequence of a Partial cDNA Derivedfrom Rice (Nippon-Bare) Showing High Homology with the ALS Gene of Maize

As a part of the Rice Genome Project conducted by the Society forTechno-innovation of Agriculture, Forestry and Fisheries, and theNational Institute of Agrobiological Sciences, partial nucleotidesequences of cDNAs of rice (Nippon-bare) had been determined and apartial nucleotide sequence database of cDNAs had already beenestablished. A cDNA clone (Accession No. C72411) which is known as anucleotide sequence of approximately 350 bp contained in this databaseshowed high homology to the ALS gene of maize. The ALS gene of maize hadbeen completely sequenced.

This cDNA clone (Accession No. C72411) was obtained from the NationalInstitute of Agrobiological Sciences, and the nucleotide sequence wasdetermined as follows. Here, the cDNA clone comprised an ALS homologgene inserted within pBluescript II SK+, and it was capable ofautonomous replication in E. coli.

First, an ALS homolog-retaining plasmid vector was transformed into E.coli (DH5α). White colonies obtained from a plate were cultured inliquid, and then plasmids were extracted from the cells by standardtechniques. Since the insert DNA had been inserted between Sal I and NotI (restriction enzymes of multi-cloning sites in the plasmid vector),the vector was digested with the two enzymes. The insert was confirmedby agarose electrophoresis. Then, the obtained ALS homolog-retainingplasmid vector was purified by standard techniques using, e.g., RNaseA,PEG and LiCl, followed by sequencing reaction using primers and an ABIBigDyeTerminator Cycle Sequencing Kit. Conditions for PCR reactionfollowed the manufacturer's protocols. Primers used herein were M13primers and synthesized primers designed from the determined nucleotidesequence. The resulting PCR product was purified by ethanolprecipitation, and then the nucleotide sequence thereof was determinedby an ABI PRISM 310 sequencer.

The ALS homolog-retaining plasmid vector is known to contain an insertDNA with a length of 1.6 kb. The obtained ALS homolog-retaining plasmidvector was digested with restriction enzymes Sal I and Not I, and thensubjected to electrophoresis. As a result, a band of approximately 3 kbpcorresponding to pBluescript II SK+ and a band of approximately 1.6 kbpcorresponding to the insert DNA fragment were detected (data not shown).The entire nucleotide sequence of the insert DNA portion was determined,and its homology to the nucleotide sequence of maize was searched. Asshown in FIGS. 18A and B, 84.7% homology was found. Since the ALShomolog was determined to be a partial cDNA of the ALS gene of the var.Nippon-bare, the insert DNA excised after digestion with Sal I and Not Iwas used as a probe. Further in FIGS. 18A and B, the first row is anucleotide sequence of the cDNA of the ALS gene of the var. Nippon-bare;the second row is that of the ALS gene of maize.

(2) Preparation of mRNA from Resistant Mutant and Wild Type

First, the resistant mutant frozen with liquid nitrogen was crushed witha mortar and pestle, and then finely crushed with a mixer for 30seconds. The crushed powder was suspended in an extraction buffer [(100mM Tris-HCl pH 9.0, 100 mM NaCl, 1 weight % SDS, 5 mM EDTA):(β-mercaptoethanol): (Tris saturated phenol)=15:3:20], and then stirredthoroughly. This solution was centrifuged at 12000×g for 15 minutes, andthen the supernatant was collected. Two hundred ml of PCI [(Trissaturated phenol):(chloroform):(isoamylalcohol)=25:24:1] was added tothe supernatant, shaken at 4° C. for 10 minutes, centrifuged at 12000×gfor 15 minutes, and then the supernatant was collected. The procedurewas repeated twice. A 1/20 volume of 5 M NaCl and a 2.2-fold volume ofethanol were added to the obtained supernatant, and then the mixture wasallowed to stand at −80° C. for 30 minutes. The precipitate wascollected by centrifugation at 12000×g for 5 minutes. The precipitatewas washed with 70% ethanol, dried, and then dissolved in 10 mMβ-mercaptoethanol solution. Next, the solution was centrifuged at27000×g for 10 minutes to remove insoluble fraction. A ¼ volume of 10 MLiCl was added to the solution, which was then allowed to stand on icefor 1 hour. Further, the solution was centrifuged at 27000×g for 10minutes to collect precipitate, dissolved in 4 ml of H₂O, and thenabsorbance at 260 nm was measured to find the concentration of RNA. A1/20 volume of 5 M NaCl and a 2.2-fold volume of ethanol were added tothe solution, which was then allowed to stand at −80° C. for 30 minutes.Subsequently the solution was centrifuged at 27000×g for 10 minutes tocollect the precipitate, followed by washing with 70% ethanol, anddrying. The resulting product was dissolved in an appropriate amount ofH₂O to obtain a total RNA solution. Here, centrifugation was performedat 4° C.

mRNA was separated and purified from total RNA by the following method.A 2× binding buffer (20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1 M NaCl) in avolume equivalent to that of the extracted total RNA solution was addedto the extracted total RNA solution. A column filled with 0.1 g of oligodT cellulose (Amersham Bioscience) was washed with a 1× binding buffer,and then the total RNA solution was applied to the column. After thecolumn was washed with a 1× binding buffer, an elution buffer (10 mMTris-HCl (pH 7.5), 5 mM EDTA) was applied, and the eluate collected 0.5ml at a time. Fractions that had passed through the column were appliedto another oligo dT cellulose (Amersham Bioscience) column, and treatedin the same manner. After the concentration of eluted mRNA wascalculated based on the absorbance of each fraction, a 1/10 volume of 10M LiCl and a 2.5-fold volume of ethanol were added to the products, andthen the mixtures were allowed to stand at −80° C. for 30 minutes. Next,the mixtures were centrifuged and the precipitated fractions were dried,and dissolved in 100 μl of H₂O. The thus obtained mRNA was subjected tosize fractionation by sucrose density gradient centrifugation.

The separated and purified mRNA was applied to a centrifuge tube withdensity gradient given by a 25% sucrose solution and 5% sucrosesolution, and then ultracentrifuged at 27000 rpm for 15 hours at 4° C.using a swing rotor. After centrifugation, 0.5 ml of each fraction wascollected in order of density gradient. Absorbance of each fraction wasmeasured, the concentration of the collected mRNA was calculated, andthe presence of ALS mRNA was confirmed by hybridization using an ECL kit(ECL direct nucleic acid labeling and detection system, AmershainBioscience). Hybridization was performed using a probe prepared in (1)above at 42° C. for 16 hours. After hybridization, washing at 42° C. for5 minutes was performed twice using a primary washing buffer providedwith the kit, and then washing at 42° C. for 5 minutes was performedonce using 2×SSC solution. The washed film was wrapped with atransparent plastic film to keep it immersed in an attached luminousreagent provided with the kit, and then exposed to an X-ray film.

When Sr line was used as the resistant mutant, approximately 35 mg oftotal RNA and approximately 4 mg of mRNA could be extracted by the aboveprocedures. Further, in sucrose density gradient centrifugation, ahybridization-positive spot was found for a fraction expected to bepositive.

When the wild type was used, approximately 95 mg of total RNA wasextracted in addition to approximately 7 mg of mRNA. When mRNA wasextracted from the wild type, the above method was applied except thatthe wild type was used instead of the resistant mutant.

(3) Construction of cDNA Libraries derived from Resistant Mutant andWild Type

Using 2 μg of mRNA purified in (2) above and a cDNA synthesis kit(Amersham Bioscience), cDNA was synthesized, so that a cDNA libraryderived from the resistant mutant was constructed.

First, RTase provided with the kit was used for a reverse transcriptionreaction; and T4 DNA polymerase provided with the kit was used for asubsequent complementary chain elongation reaction. At the time ofcomplementary chain elongation reaction, ³²P-dCTP was added to calculatethe yield of cDNA synthesis. After an adaptor was added, the synthesizedcDNA was incorporated into λ phage by in vitro packaging method.

The adaptor added to cDNA was an Eco RI-Not I-Bam HI adaptor (TakaraShuzo). Adapters with a molar concentration 50-fold greater than that ofcDNA were added to a solution containing cDNA. Then, T4 DNA Ligase(Pharmacia) was added to the mixture followed by ligation reaction at 4°C. overnight. The reaction solution was applied to HPLC using anAsahiPak GS 710 column (Asahi Chemical Industry Co., Ltd.), followed bymonitoring of the eluate with ultraviolet rays at a wavelength of 260nm. The eluate was fractionated into 25 fractions of 0.5 ml each. Eachfraction was measured with a Cerenkov counter, and 3 to 4 fractions witha high count were collected. The 5′ terminus of the adaptor contained inthe fraction was phosphorylated using T4 polynucleotide kinase (TakaraShuzo), and then λgt 11 Eco RI arm was added to perform ligation.GigaPack Gold III (Stratagene) was added to the solution, and thenligation reaction was performed at room temperature for 2 hours. Afterreaction, 200 μl of an SM buffer and 8 μl of chloroform were added tothe reaction solution, thereby preparing a phage solution. This phagesolution was diluted 10-fold. One μl of the diluted solution wasinfected with E. coli (Y-1088), to which 0.7% top agar was added, andthen the solution was inoculated over an LB plate. The number of plaquesthat had appeared on the plate 4 to 8 hours later was counted, therebymeasuring the titer.

Synthesis of approximately 74 ng of cDNA derived from Sr line wasconfirmed by the result of DE 81 paper and Cerenkov counting. The resultof Cerenkov counting after ligation of a vector with an adaptor addedthereto revealed that approximately 22 ng of λDNA contained the insertwas obtained for Sr line. The λDNA was packaged into the phage, therebypreparing a cDNA library derived from the cells of the resistant mutant.The titer of the library solution was 16600 pfu/μl.

When a cDNA library was constructed using mRNA extracted from the wildtype according to the above-described method, it was shown thatapproximately 38 ng of cDNA derived from the wild type had beensynthesized. Further, approximately 5 ng of λDNA contained the insertwas obtained for the wild type. Furthermore, the titer of the cDNAlibrary solution derived from the wild type was 18160 pfu/μl.

(4) Screening of cDNA Containing the ALS Gene

To form about 20,000 plaques on plates, the library solution prepared in(3) above was diluted, and then phages derived from the wild type andthose derived from Sr line were separately inoculated over 10 plates,respectively. Plaques were then transferred to a nitrocellulose membrane(Schleicher & Schnell, PROTORAN BA85, pore size 0.45 μm), and thenitrocellulose membrane was immersed in a denaturation solution (0.5 MNaOH, 1.5 M NaCl), and then in a neutralization solution (1.5 M NaCl,0.5 M Tris-HCl (pH 7.5), 1 mM EDTA) for approximately 20 seconds. Excesswater was removed from the nitrocellulose membrane using a filter paper,and then the nitrocellulose membrane was baked at 80° C. for 2 hours.Here, the baking step was omitted when Hybond-N+(Amersham Biotech) wasused instead of a nitrocellulose membrane, and immobilization wasperformed with 0.4 M NaOH for 20 minutes.

The insert DNA prepared in (1) above was labeled by two types of method,RI and non-RI, and then used as a probe DNA. Labeling with RI andhybridization were performed by the following method. First,approximately 200 to 500 ng of probe DNA was thermally denatured, andthen labeled using a BcaBEST DNA labeling kit (Takara Shuzo). At thetime of this labeling reaction, a buffer, random primers and ³²P-dCTPprovided with the kit were added. Next, BcaBEST was added, followed byincubation at 65° C. for 30 minutes. Subsequently, EDTA was added tostop the reaction. The reaction solution was applied to nitrocellulosemembranes, so that 8 of the membranes contained approximately 100 ng ofprobes. Hybridization was performed at 42° C. overnight with weakshaking. After hybridization, the membranes were washed three times with2×SSC, 0.1% SDS solution, followed by exposure for about 1 hour to animaging plate of a BAS 2000 imaging analyzer (Fuji Photo Film).Following exposure, positive clones were detected using the imaginganalyzer.

Labeling with non-RI was performed by the following method. Followingthermal denaturation of approximately 200 to 500 ng of probe DNA, DNAlabeling reagent (peroxidase) and glutaraldehyde which were providedwith an ECL direct DNA/RNA labeling and detection system (AmershamBioscience) were added, followed by incubation at 37° C. In this case,the labeled probe DNA was applied to nitrocellulose membranes, so that 8of the membranes contained approximately 100 ng of the labeled probeDNA. Hybridization was performed at 42° C. overnight with weak shaking.After hybridization, the membrane was washed three times with a primarywashing buffer at room temperature for 10 minutes, and then once with2×SSC at room temperature for 10 minutes. The membrane was immersed in aluminous solution provided with the ECL kit, and then exposed to anX-ray film for 30 minutes to 3 hours.

Positive phages obtained by hybridization (primary screening) werescraped off together with top agar using a sterile toothpick, and thensuspended in 200 μl of SM buffer, thereby obtaining a phage solution.Phage solutions of each clone were appropriately diluted, infected withE. coli strain Y-1088, and then inoculated over LB plates. Using thesenewly prepared plates, hybridization (secondary screening) was performedsimilarly. Positive phages were suspended in 200 μl of a SM buffer,thereby obtaining single phages. If no single phage was isolated bysecondary screening, another dilution was performed, followed byinoculation over LB plates. Subsequently, hybridization (the thirdscreening) was performed, so that single phages were obtained.

Next, λDNA was prepared from the single phages by the following methods.λ phages collected with a bamboo brochette or a toothpick from plaquesof positive clones were inoculated in 200 μl of a 2×YT medium(containing 10 mM MgCl₂ and 0.2% maltose) containing 5 μl of asuspension of fresh host E. coli (Y1088). The product was allowed tostand and incubated at 42° C. overnight. Then, the medium was inoculatedagain in 1 ml of a 2×YT medium (containing 10 mM MgCl₂ and 0.2% maltose)containing 25 μl of a suspension of host E. coli (Y1088), and thenshake-cultured overnight (these steps compose a pre-culturing process).The pre-cultured solution (10 to 50 μl) was inoculated in 12 ml of 2×YTmedium containing 10 mM MgCl₂ and 0.5 ml of E. coli Y1088 suspension.Then, incubation was performed at 42° C. overnight with relativelystrong shaking, until turbidity increased after lysis. After culturing,50 μl of chloroform and 1.2 ml of 5 M NaCl were added, and thenincubation was performed at 42° C. for 10 minutes while shaking. Theproduct was centrifuged at 27000×g for 10 minutes, and then thesupernatant was newly transferred to a centrifugation tube. Five ml of50% PEG was added to the supernatant, and then incubated on ice for 1hour or more. The product was centrifuged at 27000×g for 10 minutes, andthen the supernatant was discarded. Next, another centrifugation wasperformed at 27000×g, and then the liquid portion was discarded. Theprecipitated fraction was suspended in 300 μl of a 30 mM Trishydrochloric acid buffer (pH 7.5) containing 4 μg of DNase I, 20 μg ofRNase A and 10 mM MgCl₂. The suspension was transferred to a 1.5 mltube. After incubation of the suspension at 37° C. for 30 minutes, 7.5μl of 20% SDS, 3 μl of proteinase K (10 mg/ml), and 12 μl of 0.5 M EDTAwere added to the suspension, followed by further incubation at 55° C.for 15 minutes. Subsequently, 150 μl of phenol was added to the product,and then stirred vigorously. Then the mixture was centrifuged at 15000rpm for 3 minutes using a TOMY Microcentrifuge MR-150 (TOMY DIGITALBIOLOGY), and an aqueous layer was collected. 800 μl of ethyl ether (towhich distilled water had been added to remove peroxide) was added tothe collected aqueous layer. The mixture was stirred vigorously, andthen centrifuged at 15000 rpm for 10 seconds and the ether layer wasdiscarded. After the ether extraction step was repeated, ether remainingin the aqueous layer was removed with nitrogen gas. Thirty μl of 5 MNaCl and 875 μl of ethanol were added to the aqueous layer, so thatprecipitated λDNA was rapidly collected. The collected λDNA was rinsedwith approximately 1 ml of 70% ethanol, and then dried under reducedpressure for approximately 1 minute, thereby removing ethanol. Theproduct was dissolved in 20 μl to 50 μl of a TE buffer (pH 8.0), therebypreparing a λDNA solution.

Subcloning and sequencing of the insert DNA in the obtained λDNA wereperformed by the following method. The obtained λDNA solution (1 μl) wasdigested with Not I so as to excise the insert DNA. The composition of areaction solution (for cleavage reaction) followed the procedure in themanual attached to the restriction enzyme. After reaction at 37° C. forapproximately 2 hours, the insert size was confirmed by electrophoresisusing 1% agarose gel. λDNA (10 μl to 20 μl) containing the insert DNAwas digested with Not I, so as to excise the insert DNA. The insert DNAwas separated using agarose gel for apportioning, the corresponding bandwas cleaved from the gel, and then the insert DNA was purified bystandard techniques. The insert DNA was mixed with a vector followingBAP treatment (dephosphorylation using alkaline phosphatase derived froma shrimp) at molar ratio of 1:1, followed by ligation reaction with T4DNA ligase at 16° C. for 2 hours or more. Here, since the insert DNAcleaved with Not I was used as material, BAP treatment was performed forvectors cleaved with Not I. Following ligation, part of the solution wasmixed with competent cells (DH5α), and then allowed to stand on ice for30 minutes. Next, the mixture was subjected to heat shock at 42° C. for30 seconds, and then allowed to stand on ice again for 2 minutes. Then,SOC was added to the mixture, incubated at 37° C. for 1 hour, inoculatedover a LB medium plate on which a mixture of 100 μl of 2×YT (containing50 μg/ml ampicillin), 30 μl of 3% X-Gal and 3 μl of 1 M IPTG had beenpreviously added uniformly, and then cultured at 37° C. for 10 hours ormore. The transformed white colonies were each inoculated on 2 ml of anLB medium containing ampicillin or a 2×YT medium, and then cultured at37° C. overnight. From the culture solution, plasmids were prepared bystandard techniques and dissolved in H₂O. The DNA concentration thereofwas quantified, and then the plasmids were subjected to PCR reaction forsequencing. PCR reaction and sequencing were performed by methodsdescribed above.

As a result of the above experiment, the ALS cDNA with an incompletelength of approximately 2.2 kb was obtained from the culture cells ofeach wild type and Sr line. Since an Sma I site was present at aposition approximately 250 bp from the 5′ side of the DNA, a new probewas prepared by the following method. pBluescript II SK+ retaining theALS cDNA with an incomplete length of approximately 2.2 kbp derived fromthe wild type was amplified with host E. coli JM109, and then plasmidswere extracted using an automated isolation system (KURABO PI-100). Theplasmid was directly digested with Sma I. The generated fragment ofapproximately 250 bp was separated and purified by 1% agaroseelectrophoresis, and then the concentration was calculated, therebypreparing a probe. Using the probe, the library was screened again bythe above method employing the above RI. λDNA was prepared from the thusobtained single phages, the λDNA solution (1 μl) was digested with EcoRI, and then size was confirmed by electrophoresis, followed byimmobilization onto a nitrocellulose membrane. Followingelectrophoresis, the gel was immersed in 0.5 M NaOH solution containing1.5 M NaCl, and then shaken lightly for 15 minutes. The gel was thenwashed with water, immersed in 0.5 M Tris-HCl (pH 7.5) containing 3 MNaCl, and then neutralized while shaking for approximately 15 minutes.Approximately 5 thick, industrial filter papers were piled up to make abase. The base was placed in 20×SSC spread over a stainless bat.Subsequently, the neutralized gel, a nitrocellulose membrane (which hadbeen cut into a certain size, immersed in distilled water and thenimmersed in 20×SSC for another 10 minutes), and two-ply filter paperswere placed in order on the base, on which a paper towel with athickness of 3 cm to 4 cm was further placed. A glass plate and then alight weight were placed on the product, followed by blotting forapproximately 5 minutes. After confirming that no bubbles were entrappedbetween the gel and the membrane, blotting was performed forapproximately 10 minutes. Following blotting, the membrane was subjectedto UV treatment with a trans-illuminator, and then baked at 80° C. forapproximately 15 minutes to 30 minutes. After baking, hybridization(hybridization buffer composition: 5×SSPE, 0.5% SDS, 5× Denharlts, solumsperm DNA, 50% formamide) was performed with the above 250 bp probe DNAlabeled with ³²P. Radiation of the hybridized band was transferred to animaging plate, and the result was analyzed with BAS-2000. Among insertspositive in hybridization, those showing a relatively large size wereprepared in large quantity, and then sub-cloned into pBluescript II SK+that had been digested with Eco RI and then treated with BAP by theabove method. The product was transformed into E. coli (JM 105). Theobtained transformants were subjected to liquid culture, and thenplasmids were prepared by standard techniques. Thus, the nucleotidesequence was determined by the above methods.

As a result, the full-length ALS cDNA gene could be obtained from theculture cells of each wild type and Sr line. The results of homologycomparisons between the wild type and the mutant ALS genes are shown inFIGS. 19A; B and C. As shown in FIGS. 19A, B, and C, compared to thewild type, 2-point mutations were observed in Sr line at 2 points, the1643rd and 1880^(th), from the first base A as the starting point of thetranscription initiation codon ATG. In Sr line, the 1643rd G in the wildtype was mutated to T (denoted as G1643T), and the 1880th G in the wildtype was mutated to T (denoted as G1880T). When converted into aminoacids, these mutations indicated that the mutant ALS protein of Sr linehad an amino acid sequence wherein the 548th tryptophan in the wild typeALS protein was mutated to leucine (that is, “W548L mutation”), and the627th serine in the wild type ALS protein was mutated to isoleucine(that is, “S627I mutation”).

(5) Subcloning of the Wild Type ALS cDNA Cloned into pBluescript II SK+into pGEX 2T

After the pBluescript II SK+ plasmid having the full-length wild typeALS cDNA obtained in (4) above incorporated therein was digested withEco RI, cDNA containing the wild type ALS gene was excised. Then, thecDNA was incorporated into Eco RI site of pGEX-2T (Amersham Bioscience),which is an E. coli expression vector. Hereinafter, an expression vectorhaving the full-length wild type ALS cDNA incorporated into the Eco RIsite of pGEX-2T is referred to as “pGEX-2T(ALS-wild).” pGEX-2T(ALS-wild)was transformed into E. coli (JM 109). Colonies obtained bytransformation were liquid-cultured, plasmids were extracted, and thenthe insertion direction of insert DNA was confirmed by sequencing. Thus,E. coli (JM109) transformed with pGEX-2T(ALS-wild) was prepared.

EXAMPLE 4 Elucidation of Mutation Sites in ALS Gene of PC HerbicideResistant Rice Culture Cell

(1) Extraction of Genomic DNA from Resistant Mutant (Strains of Sr, Rb,Vg, and Ga Lines)

Using a plant DNA extraction kit ISOPLANT II (Nippon Gene), genomic DNAwas extracted from 0.1 g of cultured cells of each of Sr, Rb, Vg and Galines according to the protocols attached to the kit. After genomic DNAwas extracted using the above kit, RNA was denatured and removed usingRNase A. Then, agarose gel electrophoresis was performed, therebyconfirming the genomic DNA.

(2) PCR of ALS Gene using Genomic DNA as Template

PCR was performed using each genomic DNA as a template, and a primer“ALS-Rsp3” and a primer “4-83-3,” as shown below. PCR was performedusing Ready to Go PCR Beads (Amersham Bioscience) at a final volume of25 μl. The reaction was performed for 40 cycles, each cycle conditionconsisting of an initial denaturation step at 94° C. for 5 minutes,followed by a denaturation step at 94° C. for 30 seconds, annealing stepat 55° C. for 1 minute, and elongation step at 72° C. for 2 minutes. Inaddition, the elongation step in the final cycle was performed at 72° C.for 9 minutes.

Next, the PCR reaction solution was subjected to 2% agarose gelelectrophoresis (100V, 1×TBE buffer). Gels containing PCR products wereexcised, and then excised gels were cut into small fragments. Theobtained gel fragments were rinsed twice or three times with sterile ionexchanged water, 500 μl of sterile ion exchanged water was added, andthen freezing and dissolving was repeated three times. Thus, the PCRproduct could be eluted in water.

Next, PCR was performed again using the eluate in which the PCR producthad been dissolved. Specifically, this PCR was performed at a finalvolume of 100 μl using the PCR product contained in the solution as atemplate, and the same primer set or nested primers. After reaction, thereaction solution was subjected to agarose gel electrophoresis (1%) forapportioning. Gels containing target bands were excised, and thenpurified using a GFX PCR DNA & Gel Band Purification Kit (AmershamBioscience). Finally, the PCR product was eluted using 75 μl of steriledeionized water.

(3) Sequencing

Sequence reaction was performed using the DNA fragment amplified by PCRas a template and ABI PRISM BigDye ver.2 (Applied Biosystem). Forsequence reaction, 11 μl of the template DNA, 1 μl of the primer (3.2pmol/μl) and 8 μl of pre-mix was mixed, therefore the total volume was20 μl. The sequence reaction was performed for 40 cycles, each cyclecondition consisting of an initial denaturation step at 96° C. for 5minutes, followed by a denaturation step at 96° C. for 5 seconds,annealing step at 50° C. for 5 seconds, and elongation step at 60° C.for 4 minutes. In addition, the elongation step of the final cycle wasperformed at 60° C. for 9 minutes. After sequence reaction, fluorescentnucleotides in the reaction solution were removed by gel filtrationusing AutoSeq G-50 column (Amersham Biotech). Then the nucleotidesequences were read using ABI PRISM 310 DNA sequencer.

(4) Names of Primers and Nucleotide Sequences used Herein

Names, nucleotide sequences and the like of primers used in (2) aboveand of primers used in the following examples are listed in Table 6.TABLE 6 Corresponding Number Name Nucleotide sequence Direction ALS siteof bases ALS-Rsp1 5′-GCTCTGCTACAACAGAGCACA-3′ sense 1192-1212 21 merALS-Rsp2 5′-AGTCCTGCCATCACCATCCAG-3′ antisense 1906-1926 21 mer ALS-Rsp35′-CTGGGACACCTCGATGAAT-3′ sense 720-738 19 mer ALS-Rsp45′-CAACAAACCAGCGCAATTCGTCACC-3′ antisense 862-886 25 mer ALS-Rsp65′-CATCACCAACCACCTCTT-3′ sense 327-344 18 mer ALS-Rsp75′-AACTGGGATACCAGTCAGCTC-3′ antisense 886-906 21 mer ALS-RspA5′-TGTGCTTGGTGATGGA-3′ antisense 571-586 16 mer ALS-RspB5′-TCAAGGACATGATCCTGGATGG-3′ sense 1913-1944 16 mer ALS-RspC5′-CAGCGACGTGTTCGCCTA-3′ sense 258-275 16 mer ALS-RspD5′-CCACCGACATAGAGAATC-3′ antisense 828-845 18 mer ALS-RspF5′-ACACGGACTGCAGGAATA-3′ antisense 1749-1766 18 mer ALS-RspE5′-TTACAAGGCGAATAGGGC-3′ sense 1656-1673 18 mer 3-1-15′-GCATCTTCTTGATGGCG-3′ antisense 1791-1807 17 mer 3-1-25′-ATGCATGGCACGGTGTAC-3′ sense 973-990 18 mer 3-1-35′-GATTGCCTCACCTTTCG-3′ antisense 1346-1362 17 mer 3-1-45′-AGGTGTCACAGTTGTTG-3′ sense 1506-1522 17 mer 4-83-15′-AGAGGTGGTTGGTGATG-3′ antisense 327-343 17 mer 4-83-35′-GCTTTGCCAACATACAG-3′ antisense 1944-1960 17 mer 4-83-105′-CAGCCCAAATCCCATTG-3′ antisense 1457-1473 17 mer 4-83-155′-ATGTACCCTGGTAGATTC-3′ antisense 735-752 18 mer ALS-DG75′-GTITT(CT)GCITA(CT)CCIGG(ACGT)GG-3′ sense 265-284 20 mer

In Table 6, the corresponding ALS site is the number of a correspondingbase when a transcription initiation codon (ATG) is the starting point.In addition, the nucleotide sequence of ALS-Rsp1 is shown in SEQ ID NO:9, the nucleotide sequence of ALS-Rsp2 is shown in SEQ ID NO: 10, thenucleotide sequence of ALS-Rsp3 is shown in SEQ ID NO: 11, thenucleotide sequence of ALS-Rsp4 is shown in SEQ ID NO: 12, thenucleotide sequence of ALS-Rsp6 is shown in SEQ ID NO: 13, thenucleotide sequence of ALS-Rsp7 is shown in SEQ ID NO: 14, thenucleotide sequence of ALS-RspA is shown in SEQ ID NO: 15, thenucleotide sequence of ALS-RspB is shown in SEQ ID NO: 16, thenucleotide sequence of ALS-RspC is shown in SEQ ID NO: 17, thenucleotide sequence of ALS-RspD is shown in SEQ ID NO: 18, thenucleotide sequence of ALS-RspF is shown in SEQ ID NO: 19, thenucleotide sequence of ALS-RspE is shown in SEQ ID NO: 20, thenucleotide sequence of 3-1-1 is shown in SEQ ID NO: 21, the nucleotidesequence of 3-1-2 is shown in SEQ ID NO: 22, the nucleotide sequence of3-1-3 is shown in SEQ ID NO: 23, the nucleotide sequence of 3-1-4 isshown in SEQ ID NO: 24, the nucleotide sequence of 4-83-1 is shown inSEQ ID NO: 25, the nucleotide sequence of 4-83-3 is shown in SEQ ID NO:26, the nucleotide sequence of 4-83-10 is shown in SEQ ID NO: 27, thenucleotide sequence of 4-83-15 is shown in SEQ ID NO: 28, and thenucleotide sequence of ALS-DG7 is shown in SEQ ID NO: 29.

(5) Mutations in each Line Revealed as a Result of Sequencing

As a result of analysis of nucleotide sequences determined in (3) above,mutations in Rb, Vg, Ga, and Sr lines were revealed. The mutated pointsof each line are listed in Table 7. TABLE 7 Mutant base Mutant aminoC512A C514A G1643T G1880T acid P171H R172S W548L S627I Rb line homohetero Vg line hetero Ga line homo or hetero homo or hetero hetero Srline hetero hetero

As shown in Table 7, in the nucleotide sequence of Rb line strain, the512^(nd) C was mutated to A (homo), and the 1643^(rd) G was mutated to T(hetero). This means that at the amino acid level, the 171^(st) prolineand the 548^(th) tryptophan (W) were mutated to histidine (H) andleucine (L), respectively. In the nucleotide sequence of Vg line strain,the 1643^(rd) G was mutated to T (hetero), suggesting that at the aminoacid level, the 548^(th) tryptophan (W) was mutated to leucine (L). Inthe nucleotide sequence of Ga line strain, the 512^(nd) and 514^(th) Cwere mutated to A (homo or hetero) (these types differed depending onthe PCR product obtained), and the 1643^(rd) G was mutated to T(hetero). This means that at the amino acid level, the 171^(st) proline(P), 172^(nd) arginine (R) and 548^(th) tryptophan (W) were mutated tohistidine (H), serine (S) and leucine (L), respectively. Further, in thenucleotide sequence of Sr line strain, the 1643^(rd) and 1880^(th) Gwere mutated to T (hetero).

When ALS genes were screened and isolated from the cDNA library of Srline strain by the above method, not only a 2-point mutant gene, butalso a gene of the wild type was isolated. Thus, it was assumed that atthe genomic DNA level, heterologous mutation had occurred, and theresults obtained by genome PCR also supported this assumption.

As described above, in all the resistant mutants, the 548^(th)tryptophan (W) was mutated to leucine (L) (hetero), and Vg line had thismutation only. As described above, Vg line strain showed sensitivity upto 10 μM bispyribac-sodium, and Sr, Rb and Ga line strains showed thesame up to 100 μM bispyribac-sodium. Accordingly, it was suggested thatthe acquisition of resistance started from Vg line and branched intoother lines and mutated, as the intensity of the selection pressureincreased.

EXAMPLE 5 Synthesis of ALS cDNAs Independently having G1643T(W548L)Mutation or G1880T(S627I) Mutation, Construction of pGEX 2T Retainingthe ALS cDNAs, and Transformation of E. coli using the Vector

First, synthesis of ALS cDNAs independently having G1643T(W548L)mutation or G1880T(S627I) mutation, and construction of pGEX 2Tretaining the ALS cDNAs are described using FIG. 20.

PCR was performed at a final reaction volume of 100 μl using 1 μl (585ng/μl and 554 ng/μl, respectively) of pBluescript II SK+ (ALS-2 pointmutant) or pBluescript II SK+ (ALS-wild) as a template, and 1 μl of LATaq DNA polymerase (Takara). The reaction was performed for 25 cycles,each cycle condition consisting of 95° C. for 30 seconds, 55° C. for 30seconds and 72° C. for 2 minutes. Further, pBluescript II SK+ (ALS-2point mutant) contained 2-point mutant ALS gene, G1643T(W548L) andG1880T(S627I). pBluescript II SK+(ALS-wild) contained the wild type ALSgene having no mutation. For the PCR, a combination of ALS-Rsp6 andALS-RspF primers and a combination of ALS-RspE and M13R primers wereused. Names of fragments amplified using ALS genes as a template and thegiven combination of primers are listed in Table 8. In addition, primerM13R is an antisense primer in the vicinity of T3 promoter ofpBluescript II SK+. Further, the nucleotide sequence of M13R is5′-GGAAACAGCTATGACCATG-3′ (SEQ ID NO: 30). TABLE 8 pBluescript IISK+(ALS-2 pBluescript II point mutant) SK+(ALS-wild) ALS-Rsp6 PCR-1PCR-3 ALS-RspF ALS-RspE PCR-2 PCR-4 M13R

PCR-1, PCR-2, PCR-3 and PCR-4 obtained by PCR were respectivelysubjected to agarose gel electrophoresis for separation, and then theproducts were collected in a manner similar to the above method from theagarose gel, and then the products were eluted with 50 μl of sterilizedwater.

Next, a set of PCR-1 and PCR-4, and a set of PCR-2 and PCR-3 weresubjected to SPR (self polymerase reaction). SPR was performed by adding23.5 μl of the set of PCR-1 and PCR-4, or the set of PCR-2 and PCR-3 and1 μl of LA Taq DNA polymerase to a final volume of 75 μl, and byperforming 25 times a cycle consisting of a denaturation step at 95° C.for 1 minute, annealing step at 55° C. for 30 seconds, and elongationstep at 72° C. for 2 minutes. DNA fragments obtained by SPR using theset of PCR-1 and PCR-4 was regarded as SPR-1, and DNA fragments obtainedby SPR using the set of PCR-2 and PCR-3 as SPR-2.

Further, in this example, to secure a sufficient amount of SPR-1 and ofSPR-2, PCR was respectively performed at a final reaction volume of 100μl using purified SPR-1 or SPR-2 as a template, ALS-Rsp6 and M13R, andLA Taq DNA polymerase again. PCR in this case was performed by repeating25 times a cycle consisting of a denaturation step at 95° C. for 30seconds, annealing step at 55° C. for 30 seconds and elongation step at72° C. for 2 minutes. After PCR, the reaction solution was subjected toagarose gel electrophoresis. An approximately 2 kbp single band (PCRproduct) was collected from agarose gel, and then eluted with 100 μl ofsterilized water.

Next, SPR-1 and SPR-2 amplified by PCR were respectively digested withAcc I and Eco RI, thereby obtaining SPR-1 (Acc I/Eco RI-digestedfragment) and SPR-2 (Acc I/Eco RI-digested fragment). Specifically, 50μl of the sterilized water (100 μl in total) containing PCR productdissolved therein was mixed with 1 μl of Acc 1 (12 u/μl) and 1 μl of EcoRI (12 u/μl) in the presence of 10× M buffer (Takara), followed byincubation at a final volume of 60 μl at 37° C. for 1 hour. Afterwards,the total volume of the reaction solution was subjected to agarose gelelectrophoresis, and then a target 1.5 kbp fragment was collected usinga GFX PCR and Gel Purification Kit. The collected 1.5 kbp fragment waseluted with 50 μl of sterilized water, so that a solution containingSPR-1 (Acc I/Eco RI-digested fragment) and a solution containing SPR-2(Acc I/Eco RI-digested fragment) were prepared.

Meanwhile, 150 μl of a protein expression vector having the wild typeALS gene incorporated therein, pGEX-2T(ALS-wild) plasmid (concentrationof approximately 50 ng/μl), was mixed with 1 μl of Acc I (12 u/μl,Takara) in the presence of 10× M buffer, followed by incubation at 37°C. for 2 hours. After reaction, a linear 7.2 kbp band was confirmed by1% agarose gel electrophoresis. According to the protocols of GFX PCRand Gel Purification Kit, DNA corresponding to the 7.2 kbp band wascollected from the agarose gel, and then the product was eluted with 180μl of sterilized water. 89 μl of the eluted product was mixed with 10 μlof 10×H buffer (Takara) and 1 μl of Eco RI (12 u/μl), and then allowedto react at 37° C. for 1 minute, thereby partially digesting the thuscollected DNA with Eco RI. After reaction, 10×loading buffer was added,and then 1.5% agarose gel electrophoresis was performed. 4.9 kbp, 5.7kbp, and 6.5 kbp bands, and a 7.2 kbp band that was not cleaved at allappeared separately, and then the target 5.7 kbp band was excised fromthe gel. An approximately 5.7 kbp DNA fragment contained in the excisedgel was collected using GFX PCR and Gel Purification Kit, and then theproduct was eluted with 50 μl of sterilized water.

Subsequently, 3 μl of fragments digested with Acc I and partiallydigested with Eco RI of the thus obtained pGEX-2T(ALS-wild) and 3 μl ofSPR-1 (Acc I/Eco RI-digested fragment) or SPR-2 (Acc I/Eco RI-digestedfragment) were respectively allowed to react in 6 μl of Takara ligationbuffer (ver.2, solution I) at 16° C. overnight.

Then, the reaction solution was transformed into E. coli competent cells(strain JM109, Takara) according to the protocols attached thereto. Thecells were inoculated on LB medium containing 50 ppm of ampicillin, andthen incubated at 37° C. overnight. As a result, several of the coloniesthat appeared were selected. PCR was directly performed using thecolonies as a template, and the set of ALS-RspE described in Table 6 andPGEX-3 (5′-CCGGGAGCTGCATGTGTCAGAGG-3′: SEQ ID NO: 31), the set of PGEX-5(5′-GGGCTGGCAAGCCACGTTTGGTG-3′: SEQ ID NO: 32) and PGEX-3, and the setof PGEX-5 and ALS-RspA described in Table 6. In addition, PGEX-3 had asequence the same as a part of an antisense strand located on the 3′side of pGEX-2T used as a vector. PGEX-5 had a sequence the same as apart of a sense strand located on the 5′ side of pGEX-2T used as avector. As the reaction condition for the ALS-RspE/PGEX-3 set, each 1 μMprimer and 1 PCR bead were dissolved in a total volume of 25 μl, andreaction was performed by repeating 40 times a cycle consisting of adenaturation step at 95° C. for 30 seconds, annealing step at 55° C. for1 minute, and elongation step at 72° C. for 2 minutes. In the case ofthe PGEX-5/PGEX-3 set and PGEX-5/ALS-RspA set, DMSO with a finalconcentration of 5% was further added to the above solution, because ofthe presence, at an upstream portion, of a region having approximately75% of GC content. As a result of this PCR, insertion of a desiredinsert was confirmed.

A colony for which the insertion of a desired insert had been confirmedwas picked up, and then shake-cultured in LB liquid medium (3 ml each,10 medias) containing 50 ppm of ampicillin at 37° C. for 12 hours. Afterculturing, plasmids were extracted (400 to 500 μl) from the media usinga plasmid extraction system (TOMY, DP-480), and then concentrated toapproximately 200 μl by centrifugation. Then, the concentrate waspurified and desalted using GFX PCR and Gel Purification Kit, and thenfinally eluted with approximately 130 μl of sterilized water.

Sequence reaction was performed using ABI PRISM BigDye ver. 2 for theseplasmids, so that the nucleotide sequence of the insert in the plasmidwas analyzed. For sequence reaction, the reaction solution was preparedto have a total volume of 20 μl by mixing 11 μl of template DNA, 1 μl ofprimer (3.2 pmol/μl) and 8 μl of pre-mix. The sequence reaction wasperformed for 40 cycles, each cycle condition consisting of an initialdenaturation step at 96° C. for 5 minutes, denaturation step at 96° C.for 5 seconds, annealing step at 50° C. for 5 seconds, and elongationstep at 60° C. for 4 minutes, and the elongation step of the final cyclewas performed at 60° for 9 minutes. After sequence reaction, fluorescentnucleotides in the reaction solution were removed by gel filtrationusing AutoSeq G-50 column, and then the nucleotide sequence wasdetermined using ABI PRISM 310 DNA sequencer.

In addition, for sequence reaction, of the primers described in Table 6,PGEX-5, ALS-RspC, ALS-Rsp3, ALS-Rsp1, 3-1-4 and ALS-RspB were used assense primers, and 4-83-3, PGEX-3, ALSRsp2, 4-83-10 and ALS-Rsp7 wereused as antisense primers.

As a result of analysis, it was confirmed that pGEX 2T vector comprisingthe mutant ALS gene with W548L mutation (described as “pGEX 2T(ALS-W548Lmutant)” in FIG. 20) and pGEX 2T vector comprising the mutant ALS genewith S627I mutation (described as “pGEX 2T(ALS-S627I mutant)” in FIG.20) were obtained. Subsequently, E. coli was transformed with these pGEX2T(ALS-W548L mutant) and pGEX 2T(ALS-S627I mutant).

EXAMPLE 6 Synthesis of ALS cDNAs Independently having C512A (P171H)Mutation Found by Genome PCR for Rb Line or C514A (R172S) Mutation Foundby Genome PCR for Ga Line, Construction of pGEX 2T Retaining the ALScDNAs, and Transformation of E. coli with this Vector

First, the synthesis of ALS cDNAs independently having C512A (P171H)mutation and C514A (R172S) mutation, and construction of pGEX 2Tretaining the ALS cDNAs are described using FIGS. 21 and 22.

To obtain C512A (P171H) mutant DNA fragment, PCR was performed using thegenomic DNA of Rb line as a template and a primer set of ALS-Rsp6 andALS-Rsp4 described in Table 6. Specifically, PCR was performed usingReady to Go PCR Beads by adding 5 μl of the template genomic DNA and 1μl of each primer (25 pmol/μl) to a final volume of 25 μl. The reactioncondition consisted of an initial denaturation step at 95° C. for 5minutes, followed by a cycle (repeated 40 times) of a denaturation stepat 95° C. for 30 seconds, annealing step at 55° C. for 1 minute, andelongation step at 72° C. for 2 minutes. In addition, the elongationstep of the final cycle was performed at 72° C. for 9 minutes.

After PCR reaction, the reaction solution was subjected to 2% agarosegel electrophoresis, a band of the PCR product (described as “PCR-5” inFIG. 21) was excised from agarose gel, and then purified using GFX PCRDNA & Gel Band Purification Kit. Next, the purified PCR-5 wasincorporated into pT7Blue T-vector (Novagen), the vector (TA cloningvector) for cloning PCR product. Specifically, 1 μl of the purified PCRproduct was mixed with 1 μl of pT7 Blue T-vector (50 ng/μl), 3 μl ofsterile deionized water and 5 μl of ligation buffer (ver 2, solution I,Takara Shuzo), and then allowed to react overnight at 16° C.

After reaction, the total volume of the reaction solution wastransformed into E. coli (strain JM109) according to standard methods.After culturing of E. coli on LB solid medium containing 50 ppm ofampicillin, the colonies having a target sequence was selected from thesingle colonies that appeared on the medium in a manner similar toExample 5. The selected single colonies were shake-cultured in LB liquidculture solution (3 ml, 10 media) containing 50 ppm of ampicillin at 37°C. for 12 hours. After culturing, plasmids were extracted (400 to 500μl) using a plasmid extraction system (TOMY, DP-480). The plasmids wereconcentrated to approximately 200 μl by centrifugation, purified anddesalted using GFX PCR and Gel Purification Kit, and then eluted withapproximately 80 μl of sterilized water.

Fifty μl of the eluate was mixed with 10 μl of Acc I (12 u/μl) and 1 μlof Sma I (10 u/μl) in the presence of 10 μl of 10×T buffer and 10 μl of0.1% BSA to bring to a total volume of 100 μl, and then the mixture wasincubated at 37° C. for 2 hours. After reaction, the reaction solutionwas subjected to agarose gel electrophoresis, a target band was excisedand collected, and then a DNA fragment was collected according to theprotocols of GFX PCR and Gel Purification Kit. Thus, C512A (P171H)mutant DNA fragment having Sma I site and Acc I site on its termini wasobtained.

On the other hand, since C514A and C512A mutations are close to eachother, a DNA fragment having C514A (R172S) mutation only cannot beobtained by PCR using the genomic DNA extracted from Gb line as atemplate. Thus, as shown in FIG. 21, a DNA fragment having C514A (R172S)mutation only was prepared using a pair of primers to which mutatedpoints had been previously introduced. That is, PCR was respectivelyperformed using as primers having mutated points introduced thereinALS-M1 (5′-CCCCAGCCGCATGATCGGCACCGACGCCTT-3′: SEQ ID NO: 33, underlinedA is a mutated point) and ALS-M2 (5′-CGGTGCCGATCATGCGGCTGGGGACCT-3′: SEQID NO: 34, underlined T is a mutated point) and as a templatepBluescript II SK+ having the wild type ALS cDNA incorporated therein;and using a primer set of ALS-Rsp6 and ALS-M2; and using a primer set ofALS-ML and ALS-Rsp4. In addition, complementary portions are thenucleotide sequence (1^(st) to 23^(rd) nucleotides) of ALS-M1 and that(1^(st) to 23^(rd) nucleotides) of ALS-M2. When the primer set ofALS-Rsp6 and ALS-M2 were used, a DNA fragment described as “PCR-6” inFIG. 21 was amplified, and when the primer set of ALS-M1 and ALS-Rsp4was used, a DNA fragment described as “PCR-7” in FIG. 21 was amplified.

The reaction solution was prepared at the time of PCR by dissolving 1 μlof LA Taq DNA polymerase (5 units/μl, TAKARA), 10 μl of 10×LA buffer, 10μl of 25 mM MgCl₂, 16 μl of dNTPs (consisting of 25 mM of dATP, dGTP,dCTP and dTTP, respectively), 1 μl of template DNA, and 4 μl each ofsense and antisense primers (25 pmol/μl, respectively) to a total volumeof 100 μl. The reaction was performed by repeating 25 times a cycleconsisting of an initial denaturation step at 95° C. for 5 minutes, adenaturation step at 95° C. for 30 seconds, annealing step at 55° C. for1 minute, and elongation step at 72° C. for 2 minutes, and theelongation step in the final cycle was performed at 72° C. for 9minutes.

After reaction, the reaction solution was subjected to 1.5% agarose gelelectrophoresis for apportioning, target 213 bp (PCR-6) and 377 bp(PCR-7) bands were excised and purified using GFX PCR DNA & Gel BandPurification Kit, and then the thus generated DNA fragments wererespectively eluted with 100 μl of sterile deionized water.

Next, SPR was performed using the thus obtained PCR-6 and PCR-7. At thetime of SPR, a reaction solution was prepared to a total volume of 100μl by mixing 30 μl of the thus obtained eluate with 1 μl of LA Taq DNApolymerase (5 units/μl), 10 μl of 10×LA buffer, 10 μl of 25 mM MgCl₂,and 16 μl of dNTPs (consisting of 25 mM of dATP, dGTP, dCTP and dTTP,respectively). SPR was performed by repeating 40 times a cycleconsisting of an initial denaturation step at 95° C. for 5 minutes, adenaturation step at 95° C. for 30 seconds, annealing step at 55° C. for1 minute, and elongation step at 72° C. for 2 minutes, and theelongation step in the final cycle was performed at 72° C. for 9minutes.

After reaction, the reaction solution was subjected to agarose gel(1.5%) electrophoresis for apportioning, a target 560 bp band (describedas “SPR-3” in FIG. 21) was excised and purified using GFX PCR DNA & GelBand Purification Kit, and then the generated DNA fragment (SPR-3) waseluted with 100 μl of sterile deionized water. In a manner similar tothe above method, the eluted fragment was incorporated into pT7BlueT-vector and then transformed into E. coli (JM109). The E. coli wascultured, and then the thus extracted plasmid was digested with Acc Iand Sma I, thereby obtaining C514A (R172S) mutant DNA fragment havingSma I site and Acc I site at its termini.

Meanwhile, E. coli (strain JM109) transformed with pGEX-2T(ALS-wild),the plasmid having the wild type ALS gene incorporated therein, wasshake-cultured in LB liquid medium containing 50 ppm of ampicillin (2ml×15 media) overnight at 37° C. After the plasmid was extracted using aplasmid extraction system (DP-480), the extract (approximately 750 μl)was concentrated to approximately 200 μl using a vacuum centrifugationconcentrator. Then, the concentrate was desalted using GFX PCR DNA & GelBand Purification Kit, and then the plasmid was finally eluted with 200μl of sterile deionized water.

Next, the thus obtained plasmid, pGEX-2T(ALS-wild), was digested withAcc I. Specifically, 75 μl of the eluate was mixed with 9 μl of 10×Mbuffer, 3 μl of Acc I (12u/μl), and 3 μl of sterile deionized water, andthen the mixture was allowed to react at 37° C. for 3 hours. Afterreaction, the reaction solution was subjected to 1.5% agarose gelelectrophoresis for apportioning, the target band was excised andcollected, and then purified using GFX PCR DNA & Gel Band PurificationKit, and then a DNA fragment was finally eluted with 100 μl of steriledeionized water.

Subsequently, pGEX-2T(ALS-wild) digested with Acc I was partiallydigested with Sma I. Specifically, 79 μl of the eluate was mixed with 10μl of 10×T buffer, 10 μl of 0.1% BSA, and 1 μl of Sma I (10u/μl) to atotal volume of 100 μl, and then the mixture was incubated at 30° C. for1 minute. In addition, since pGEX-2T(ALS-wild) contained Sma Irecognition sequences (on the multicloning site adjacent to Thrombincleavage site of pGEX-2T, 276^(th) and 430^(th) sequences of ALS gene)located at three positions separately, partial digestion was performedin a short time. After reaction, the reaction solution was subjected toagarose gel electrophoresis, a band corresponding to the plasmid whereinonly the 430^(th) Sma I recognition sequence of ALS gene had beendigested was excised and collected, and then purified using GFX PCR DNA& Gel Band Purification Kit to remove enzyme and protein. Finally, thepurified product was eluted with 50 μl of sterile deionized water. ThisAcc I-digested/Sma I partially-digested pGEX-2T-wild type ALS cDNAfragment, C512A(P171H) mutant DNA fragment having Sma I site and Acc Isite on its termini obtained by the above method, and C514A(R172S)mutant DNA fragment were ligated by a standard method. In FIG. 22, aplasmid containing a mutant ALS gene independently having onlyC512A(P171H) mutation obtained by the method is described as“pGEX-2T(ALS P171H mutant),” and a plasmid containing a mutant ALS geneindependently having only C514A(R172S) mutation is described as“pGEX-2T(ALS R172S mutant).”

After that, E. coli (strain JM 109) was transformed using a total volumeof the reaction solution. Single colonies that appeared on LB mediacontaining ampicillin were screened by PCR in a manner similar to theabove method, so that E. coli transformed with pGEX-2T(ALS P171H mutant)and E. coli transformed with pGEX-2T(ALS R172S mutant) were selected.

EXAMPLE 7 Synthesis of 2-Point Mutant (C512A(P171H)/C514A(R172S))ALScDNA, Construction of pGEX-2T Retaining the ALS cDNA, and Transformationof E. coli using this Vector

Synthesis of 2-point mutant (C512A(P 171H)/C 514A(R172S))ALS cDNA, andconstruction of pGEX-2T retaining the ALS cDNA are described using FIG.23.

2-point mutant (C512A(P171H)/C514A(R172S))ALS cDNA was synthesized byPCR using as a template the genomic DNA extracted from Ga line,according to the method described in Example 6 above. Specifically, PCRwas performed using as a template the genomic DNA extracted from Galine, and a primer set of ALS-Rsp6 and ALS-Rsp4, thereby amplifying aDNA fragment described as “PCR-8” in FIG. 23. Then, the amplified DNAfragment was ligated into pT7Blue T-vector, followed by digestion withAcc I and Sma I, thereby obtaining C512A(P171H)/C514A(A172S) mutant DNAfragment. Next, as shown in FIG. 22, Acc I-digested/Sma Ipartially-digested pGEX-2T-wild type ALS cDNA fragment andC512A(P171H)/C514A(R172S) mutant DNA were ligated by a standard method.Thus, pGEX-2T(ALS P171H, R172S mutant) was constructed. Further, similarto Example 6, E. coli transformed with pGEX-2T(ALS P 171H, R172S mutant)was prepared.

EXAMPLE 8 Synthesis of 2-Point Mutant (C512A(P 171H)/G 1643T(W548L) andC512A(P171H)/G1880T(S627I))ALS cDNA, Construction of pGEX-2T retainingthe ALS cDNA, and Transformation of E. coli with this Vector

Synthesis of 2-point mutant (C512A(P171H)/G1643T(W548L) andC512A(P171H)/G1880T(S627I))ALS cDNA, and construction of pGEX-2Tretaining the ALS cDNA are described using FIG. 24.

First, pGEX 2T(ALS-W548L mutant) obtained in Example 5 was digested withAcc I and then partially digested with Sma I according to the method ofExample 6, so as to cause deletion of a portion from the 430^(th) Sma Irecognition sequence to Acc I recognition sequence of ALS gene. Next,this product and C512A(P171H) mutant fragment prepared in Example 6 wereligated, so that a plasmid (described as pGEX-2T(ALS-P171H, W548Lmutant) in FIG. 24), containing 2-point mutant(C512A(P171H)/G1643T(W548L)) ALS cDNA was constructed.

Meanwhile, using pGEX 2T(ALS-S627I mutant) obtained in Example 5,instead of pGEX 2T(ALS-W548L mutant), a plasmid (described as“pGEX-2T(ALS-P171H, S627I mutant)” in FIG. 24) containing 2-point mutant(C512A(P171H)/G1880T(S627I)) ALS cDNA was constructed similarly.

Further, in a manner similar to the method of Example 6, E. coli wastransformed using these pGEX-2T(ALS-P171H, W548L mutant) andpGEX-2T(ALS-P 171H, S627I mutant).

EXAMPLE 9 Synthesis of 3-Point Mutant (C512A(P171H)/G1643T(W548L)/G1880T(S627I)) ALS cDNA, Construction of pGEX-2T Retaining the ALS cDNA,and Transformation of E. coli with this Vector

Synthesis of 3-point mutant (C512A(P 171H)/G1643T(W548L)/G 1880T(S627I))ALS cDNA, and construction of pGEX-2T retaining this cDNA are describedusing FIG. 25.

First, after pGEX 2T(ALS-S627I mutant) obtained in Example 5 wasdigested with Xho I, BAP treatment was performed according to a standardmethod. Next, according to the above method, a target gene fragment (onthe vector side) was separated and purified from agarose gel. Further,pGEX 2T(ALS-W548L mutant) obtained in Example 5 was digested with Xho I,and then a fragment containing the mutation was separated and purifiedfrom agarose gel according to the above method.

Next, to construct “pGEX-2T(ALS-W548L, S627I mutant)” having 2-pointmutation, G1880T(S627I) and G1643T(W548L), the obtained DNA fragmentswere respectively subjected to ligation reaction. After reaction, thetotal volume of the reaction solution was transformed into E. coli(strain JM109). Single colonies that appeared on LB media containingampicillin were screened by PCR according to the above method, and thenE. coli having a target plasmid (pGEX-2T(ALS-W548L, S627I mutant)) wasselected.

After culturing the selected E. coli, pGEX-2T(ALS-W548L, S627I mutant)was constructed according to the above method. pGEX-2T(ALS-W548L, S627Imutant) was digested with Acc I, and then partially digested with Sma I,thereby constructing pGEX-2T(ALS-W548L, S627I mutant) wherein a portionfrom the 430^(th) Sma I recognition sequence to Acc I recognitionsequence in ALS gene had been deleted. Subsequently, ligation of thispGEX-2T and C512A(P171H) mutant fragment prepared in Example 6 wasperformed, thereby constructing a plasmid containing 3-point mutant(C512A(P171H)/G1643T(W548L)/G1880T(S627I)) ALS cDNA (described as“pGEX-2T(ALS-P171H, W548L, S627I mutant” in FIG. 25).

Further, E. coli was transformed using pGEX-2T(ALS-P171H, W548L, S627Imutant) in a manner similar to the method of Example 6.

EXAMPLE 10 Expression of Mutant ALS Protein

E. coli transformed with pGEX-2T(ALS-wild) constructed in Example 3(5),E. coli transformed with pGEX-2T(ALS-W548L mutant) constructed inExample 5, E. coli transformed with pGEX-2T(ALS-S627I mutant)constructed in Example 5, E. coli transformed with pGEX-2T(ALS P171Hmutant) constructed in Example 6, E. coli transformed with pGEX-2T(ALSR172S mutant) constructed in Example 6, E. coli transformed withpGEX-2T(ALS P171H, R172S mutant) constructed in Example 7, E. colitransformed with pGEX-2T(ALS-P171H, W548L mutant) constructed in Example8; E. coli transformed with pGEX-2T(ALS-P171H, S627I mutant) constructedin Example 8, and E. coli transformed with pGEX-2T(ALS-P171H, W548L,S627I mutant) constructed in Example 9 were respectively shake-cultured(pre-culture) at 27° C. in 2 ml of LB liquid medium containingampicillin. These types of E. coli were respectively cultured in 250 mlof LB liquid medium containing ampicillin using 1 ml of the pre-culturesolution. After culturing overnight, 1 mM IPTG was added to the media,and then culturing was performed for a further 3 to 4 hours, so that theexpression of GST fusion protein was induced. In addition, the cellswere stored at −80° C. after washing.

Preparation and purification of ALS from E. coli were performed by thefollowing method. First, the pellet of the transformant E. coli storedat −80° C. was suspended in ALS extraction buffer (potassium phosphatebuffer (pH 7.5) containing 30% glycerol and 0.5 mM MgCl₂). Specifically,2.5 ml of the buffer was added to the pellet obtained from 50 ml of theculture solution. The suspension was subjected to ultrasonication (HeatSystems-Ultrasonics, Sonicator W-225R, micro chip, output control 8,interval of approximately 1 second, twice (40 seconds each)), andsubjected to centrifugation at 15000×g, 4° C. for 20 minutes, therebyobtaining the supernatant as a crude enzyme solution.

Thus, 9 types of crude enzyme solutions containing any one of GST fusionwild type ALS protein, GST fusion W548L mutant ALS protein, GST fusionS627I mutant ALS protein, GST fusion P171H mutant ALS protein, GSTfusion R172S mutant ALS protein, GST fusion P171H/R172S mutant ALSprotein, GST fusion P171H/W548L mutant ALS protein, GST fusion P171H/S6271 mutant ALS protein and GST fusion P171H/W548L/S627I mutant ALSprotein were prepared.

EXAMPLE 11 Herbicide Sensitivity of Mutant ALS Protein

Herbicide sensitivity of the wild type ALS protein and that of mutantALS protein were examined using the 9 types of crude enzyme solutionsobtained in Example 10. Herbicide sensitivity test was performedaccording to procedures almost the same as those in Example 2. However,in this example, reaction temperature was 37° C., reaction time was 30minutes, and 10 mM valine was added to the reaction solution to inhibitALS activity derived from E. coli. Further, three types of herbicides,bispyribac-sodium, pyrithiobac-sodium, and pyriminobac, were used as PCherbicides; chlorsulfuron was used as a sulfonylurea herbicide; andimazaquin was used as an imidazolinon herbicide. Before the addition ofmutant ALS protein, the solutions of these herbicides (aqueous solutionsfor bispyribac-sodium and pyrithiobac-sodium, and acetone solutions forother herbicides) at a certain concentration were added into thereaction solutions. The final concentration of acetone was 1%.

For the 9 types of crude enzyme solutions, inhibition activity bybispyribac-sodium is shown in FIGS. 26 and 27, and Table 9, inhibitionactivity by pyrithiobac-sodium is shown in Table 10, inhibition activityby pyriminobac is shown in Table 11, inhibition activity bychlorsulfuron is shown in Table 12, and inhibition activity by imazaquinis shown in Table 13.

In Tables 9 to 13, inhibition activity by each herbicide is representedby a herbicide concentration (150) which causes 50% inhibition, when 50%inhibition is obtained at a concentration tested, and is represented byinhibition % at the highest concentration among the concentrationstested, when 50% inhibition could not be obtained. Further, in Tables 9to 13, predicted RS ratio refers to the RS ratio of a mutant ALS proteinhaving multiple mutations, which is a combined RS ratio normallypredicted from each RS ratio of mutant ALS proteins independently havinga mutation. That is, the predicted RS ratio refers to a synergisticeffect normally predicted from a combined RS ratio of mutant ALSproteins independently having a mutation. Specifically, the predicted RSratio of a mutant ALS protein having multiple mutations was calculatedby selecting RS ratios (for all the mutations corresponding to themultiple mutations of this protein) of mutant ALS proteins respectivelyhaving only one of the mutations, and then multiplying the selected RSratios. When an actual RS ratio exceeds the predicted RS ratio of amutant ALS protein having multiple mutations, this protein hasresistance exceeding the synergistic effect (resistance) predicted froma combined resistance of mutant ALS proteins independently having amutation. TABLE 9 RS ratio/ pre- Predicted dicted ALS protein type I50(μM) RS ratio RS ratio RS ratio Wild type 0.0063 P171H mutant 0.055 8.7R172S mutant 0.0062 0.98 W548L mutant 3.3 520 S627I mutant 0.26 41P171H/R172S mutant 0.048 7.6 8.5 0.89 P171H/W548L mutant 5.5% in 100μM >15000 4500 >3.3 P171H/S627I mutant 23 3700 360 10 P171H/W548L/S627I1.1% in 100 μM >16000 190000 >0.084 mutant

TABLE 10 RS ratio/ Predicted predicted ALS protein type I50 (μM) RSratio RS ratio RS ratio Wild type 0.011 P171H mutant 0.037 3.4 R172Smutant 0.011 1 W548L mutant 41% in 100 μM >9100 S627I mutant 2.2 200P171H/R172S mutant 0.14 13 3.4 3.8 P171H/W548L mutant 20% in 100μM >9100 >31000 P171H/S627I mutant 9.4 850 680 1.3

TABLE 11 RS ratio/ Predicted predicted ALS protein type I50 (μM) RSratio RS ratio RS ratio Wild type 0.008 P171H mutant 0.04 5 R172S mutant0.0092 1.2 W548L mutant 36 4500 S627I mutant 22 2800 P171H/R172S mutant0.041 5.1 6 0.85 P171H/W548L mutant 11% in 100 μM >13000 23000 >0.57P171H/S627I mutant 21% in 100 μM >13000 14000 >0.93

TABLE 12 Predict- RS ratio/ ed predicted ALS protein type I50 (μM) RSratio RS ratio RS ratio Wild type 0.013 P171H mutant 1.1 85 R172S mutant0.011 0.85 W548L mutant 9.9 760 S627I mutant 0.031 2.4 P171H/R172Smutant 5.5 420 72 5.8 P171H/W548L mutant 16% in 100 μM >7700 65000 >0.18P171H/S627I mutant 9.9 760 200 3.8 P171H/W548L/S627I 30% in 500μM >38000 160000 >0.24 mutant

TABLE 13 RS ratio/ Predicted predicted ALS protein type I50 (μM) RSratio RS ratio RS ratio Wild type 2.2 P171H mutant 3.4 1.5 R172S mutant2.3 1 W548L mutant 16% in 100 μM >45 S627I mutant 15 6.8 P171H/R172Smutant 3.9 1.8 1.5 1.2 P171H/W548L mutant 13% in 100 μM >45 >68P171H/S627I mutant 71 32 10 3.2 P171H/W548L/S627I 15% in 100 μM >45 >460mutant

Data of the above Tables 9 to 13 are described below in order.

First, data of inhibition activity by bispyribac-sodium (Table 9)revealed the following:

Among mutant ALS protein coded by the 1-point mutant genes (P171H,R172S, W548L and S627I), W548L mutant ALS protein showed the highestresistance to bispyribac-sodium (RS ratio: 520). S627I mutant ALSprotein or P171H mutant ALS protein also showed high resistance (RSratio: 41 and 8.7, respectively), but R172S mutant ALS protein showedresistance only equivalent to that of wild type ALS protein (RS ratio:0.98). These results revealed that P171H mutation, W548L mutation andS627I mutation in ALS protein are mutations effective in enhancingresistance to bispyribac-sodium. Further, R172S mutation in ALS proteinwas shown to be a silent mutation.

On the other hand, among mutant ALS proteins coded by the 2-point mutantgenes, P171H/W548L mutant ALS protein showed the strongest resistance tobispyribac-sodium (5.5% inhibition in 100 μM, and RS ratio: >15000).P171H/S627I mutant ALS protein also showed strong resistance tobispyribac-sodium (RS ratio: 3700). The degree of resistance ofP171H/R172S mutant ALS protein was approximately the same as P171Hmutant ALS protein. Further, P171H/W548L/S627I mutant ALS protein codedby the 3-point mutant gene also imparted strong resistance tobispyribac-sodium (1.1% inhibition when 100 μM, and RS ratio: >15000).In addition, actual results of herbicide dose-response on which theseresults were based are shown in FIGS. 26 and 27.

For the 2-point and 3-point mutations, the predicted RS ratios andactual RS ratios were compared. RS ratios of P171H/W548L mutant ALSprotein and P171H/S627I mutant ALS protein were significantly higherthan the predicted RS ratios (the ratio of the RS ratio to the predictedRS ratio was remarkably larger than 1). These results revealed thatthese two 2-point mutant genes (the gene coding for P171H/W548L mutantALS protein, and the gene coding for P171H/S627I mutant ALS protein)impart resistance against bispyribac-sodium to ALS protein which isstronger than an additive effect predicted from the degree of eachresistance of the 1-point mutant gene.

Next, inhibition activity by pyrithiobac-sodium (Table 10) revealed thefollowing:

Among mutant ALS proteins (P171H, R172S, W548L and S627I) coded by1-point mutant genes, W548L mutant ALS protein showed the strongestresistance to pyrithiobac-sodium (41% in 100 μM, and RS ratio: >9100).S627I mutant ALS protein also showed resistance (RS ratio: 200), but thedegree of the resistance of P171H mutant ALS protein was low (RS ratio:3.4). R172S mutant ALS protein showed resistance only equivalent to thatof the wild type ALS protein (RS ratio: 0.85). These results revealedthat P171H mutation, W548L mutation and S627I mutation in ALS proteinsare effective mutations in enhancing resistance to pyrithiobac-sodium.Further, R172S mutation in ALS protein was shown to be a silentmutation.

On the other hand, among the mutant ALS proteins coded by 2-point mutantgenes, P171H/W548L mutant ALS protein imparted the strongest resistance(20% inhibition in 100 μM, and RS ratio: >9100), followed by P171H/S627Imutant ALS protein (RS ratio: 850). Unlike the data of inhibitionactivity by bispyribac-sodium shown in Table 9, in the case ofpyrithiobac-sodium, P171H/R172S mutant ALS protein showed a degree ofresistance higher than that of P171H mutant ALS protein (RS ratio: 13).Thus, it was clarified that R172S mutation, which is a silent mutationby itself, enhances the degree of resistance of P171H mutant ALSprotein.

Further, for 2-point mutant ALS proteins, when a combined RS ratiopredicted from each RS ratio of 1-point mutant ALS proteins and theactual RS ratio were compared, it was found that the RS ratio ofP171H/R172S mutant ALS protein was significantly higher than that of thepredicted RS ratio (the ratio of the actual RS ratio to the predicted RSratio was remarkably larger than 1). These results revealed thatP171H/R172S mutant ALS protein showed resistance to pyrithiobac-sodiumstronger than that predicted from the degrees of resistances of the1-point mutant genes.

Next, inhibition activity by pyriminobac (Table 11) revealed thefollowing:

Among mutant ALS proteins coded by 1-point mutant genes (P171H, R172S,W548L and S627I), W548L mutant ALS protein showed the strongestresistance to pyriminobac (RS ratio: 4500). S627I mutant ALS proteinalso imparted strong resistance (RS ratio: 2800), but the degree ofresistance of P171H mutant ALS protein was low (RS ratio: 5). R172Smutant ALS protein showed resistance only equivalent to that of the wildtype ALS protein (RS ratio: 1.2). These results revealed that P171Hmutation, W548L mutation and S627I mutation in ALS proteins aremutations effective in enhancing resistance to pyriminobac. Further,R172S mutation in ALS protein was shown to be a silent mutation.

Among the mutant ALS proteins coded by the 2-point mutant genes,P171H/W548L mutant ALS protein imparted the strongest resistance (11%inhibition in 100 μM, and RS ratio: >13000), followed by P171H/S627Imutant ALS protein (21% inhibition when 100 μM, and RS ratio: >13000).For these P171H/W548L mutant ALS and P171H/S627I mutant ALS proteins,predicted RS ratios and actual RS ratios were compared. However, itcould not be clarified whether resistance stronger than the resistancepredicted from the degrees of resistances of each 1-point mutant gene isshown.

Next, inhibition activity by chlorsulfuron (Table 12) revealed thefollowing:

Among the mutant ALS proteins coded by 1-point mutant genes (P171H,R172S, W548L and S627I), W548L mutant ALS protein showed the strongestresistance to chlorsulfuron (RS ratio: 760). P171H mutant ALS proteinshowed relatively strong resistance (RS ratio: 85), but the degree ofresistance of S627I mutant ALS protein was low (RS ratio: 2.4). R172Smutant ALS protein showed resistance only equivalent to that of the wildtype ALS protein (RS ratio: 0.85). These results revealed that P171Hmutation and W548L mutation in ALS protein are mutations effective inenhancing resistance to chlorsulfuron. Further, R172S mutation in ALSprotein was shown to be a silent mutation.

Among the mutant ALS proteins coded by 2-point mutant genes, P171H/W548Lmutant ALS protein imparted the strongest resistance (16% inhibition in100 μM, and RS ratio: >7700), followed by P171H/S627I mutant ALS protein(RS ratio: 760). Unlike the data of inhibition activity bybispyribac-sodium shown in Table 9, in the case of chlorsulfuron,P171H/R172S mutant ALS protein showed a degree of resistance (RS ratio:420) higher than that of P171H mutant ALS protein. Thus, it wasclarified that R172S mutation, which is a silent mutation by itself,enhances the degree of resistance of P171H mutant ALS protein. Further,P171H/W548L/S627I mutant ALS protein also imparted strong resistance(30% inhibition in 500 μM, and RS ratio: >38000).

For P171H/R172S mutant ALS and P171H/S627I mutant ALS proteins,predicted RS ratios and actual RS ratios were compared. For bothproteins, the actual RS ratios were significantly higher than thepredicted RS ratios. These results revealed that P171H/R172S mutant ALSprotein and P171H/S627I mutant ALS protein showed resistance tochlorsulfuron stronger than that predicted from the degrees ofresistances of each 1-point mutant gene.

Next, data of inhibition activity by Imazaquin (Table 13) revealed thefollowing:

Among the mutant ALS proteins coded by 1-point mutant genes (P171H,R172S, W548L and S627I), W548L mutant ALS protein showed the strongestresistance to imazaquin (16% in 100 μM, and RS ratio: >45). S627I mutantALS protein also showed resistance (RS ratio: 6.8), but P171H mutant ALSprotein showed almost no resistance (RS ratio: 1.5). R172S mutant ALSprotein showed resistance only equivalent to that of the wild type ALSprotein (RS ratio: 1.0). These results revealed that W548L mutation andS627I mutation in ALS protein are mutations effective in enhancingresistance to imazaquin. Further, P171H mutation and R172S mutation inALS protein were shown to be silent mutations against imazaquin.

Among the 2-point mutant genes, P171H/W548L mutant ALS protein impartedthe strongest resistance (13% inhibition in 100 μM, and RS ratio: >45),followed by P171H/S627I mutant ALS protein (RS ratio: 32). The degree ofresistance of P171H/R172S mutant ALS protein was almost the same as thatof p171H 1-point mutant gene. Further, P171H/W548L/S627I mutant ALSprotein also imparted strong resistance (15% inhibition in 100 μM, andRS ratio: >45).

For these 2-point ALS mutant proteins and 3-point ALS mutant protein,predicted RS ratios and actual RS ratios were compared. The RS ratio ofP171H/S627I mutant ALS protein was significantly higher than thepredicted RS ratio (the ratio of the actual RS ratio to the predicted RSratio was clearly larger than 1). These results revealed thatP171H/S627I mutant ALS protein showed resistance to imazaquin strongerthan that predicted from the degrees of resistances of each 1-pointmutant gene.

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention can provide a genecoding for acetolactate synthase showing good resistance to variousherbicides, an acetolactate synthase protein coded by the gene, arecombinant vector having the gene, a transformant having therecombinant vector, a plant having the gene, a method for rearing theplant, and a method for selecting a transformant cell using the gene asa selection marker.

Sequence Listing Free Text

SEQ ID NOS: 9 to 34 represent primers.

The 15^(th) n in SEQ ID NO: 29 represents a, c, g or t.

1. A gene, which codes for the following protein (a) or (b): (a) aprotein consisting of an amino acid sequence of any one of SEQ ID NOS:2, 4, 6, and 8; (b) a protein consisting of an amino acid sequencederived from the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6,and 8 by substitution, deletion or addition of at least one or moreamino acids, has resistance to a pyrimidinyl carboxy herbicide, and hasacetolactate synthase activity.
 2. An acetolactate synthase protein,which is coded by the gene of claim
 1. 3. A recombinant vector, whichhas the gene of claim
 1. 4. A transformant, which has the recombinantvector of claim
 3. 5. A plant, which has the gene of claim 1 and hasresistance to a pyrimidinyl carboxy herbicide.
 6. A method forcultivating the plant of claim 5, which comprises cultivating the plantin the presence of a pyrimidinyl carboxy herbicide.
 7. A method forselecting a transformant cell having the gene of claim 1, which uses thegene as a selection marker.
 8. A method for cultivating a plant having agene coding for acetolactate synthase, which comprises cultivating theplant in the presence of a pyrithiobac sodium herbicide and/or apyriminobac herbicide, wherein the acetolactate synthase has an aminoacid sequence in which a serine corresponding to serine at position 627of a wild-type rice acetolactate synthase is replaced by isoleucine. 9.A method for selecting a transformant cell having a gene coding foracetolactate synthase as a selection maker, which comprises cultivatingthe cell in the presence of a pyrithiobac sodium herbicide and/or apyriminobac herbicide, wherein the acetolactate synthase has an aminoacid sequence in which a serine corresponding to serine at position 627of a wild-type rice acetolactate synthase is replaced by isoleucine.